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1
Matos, Maria J., Libby Brown, Barbara Bernardim, Ana Guerreiro, Gonzalo Jiménez-Osés, and Gonçalo J. L. Bernardes. "Sequential dual site-selective protein labelling enabled by lysine modification." Bioorganic & Medicinal Chemistry 28, no. 22 (2020): 115783. http://dx.doi.org/10.1016/j.bmc.2020.115783.
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Enrique, Gil de Montes, Jiménez-Moreno Ester, L. Oliveira Bruno, et al. "Azabicyclic vinyl sulfones for residue-specific dual protein labelling." Chem. Sci. 10 (March 18, 2019): 4515–22. https://doi.org/10.1039/C9SC00125E.
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We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are moderately resistant to retro Diels–Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1–5 equiv. of reagent in NaP<sub>i</sub> pH 7.0, room temperature, 30 min). The resulting thioether-linked conjugates were stable and retained the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein could be fluorescently labelled through an inverse electron demand Diels–Alder reaction in cells to allow selective apoptosis imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine.
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Kwan, Terence T. L., Omar Boutureira, Elizabeth C. Frye, et al. "Protein modification via alkyne hydrosilylation using a substoichiometric amount of ruthenium(ii) catalyst." Chemical Science 8, no. 5 (2017): 3871–78. http://dx.doi.org/10.1039/c6sc05313k.
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The development of site-specific modification of alkyne-functionalized proteins using dimethylarylsilanes and substoichiometric or low-loading of Ru(ii) catalysts is reported. Furthermore, the resultant gem-vinylsilane can undergo further targeted chemical modifications, highlighting its potential for single-site, dual-modification applications.
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Crochet, Amanda P., Mohiuddin M. Kabir, Matthew B. Francis, and Chad D. Paavola. "Site-selective dual modification of periplasmic binding proteins for sensing applications." Biosensors and Bioelectronics 26, no. 1 (2010): 55–61. http://dx.doi.org/10.1016/j.bios.2010.05.012.
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Nathani, Ramiz I., Paul Moody, Vijay Chudasama, Mark E. B. Smith, Richard J. Fitzmaurice, and Stephen Caddick. "A novel approach to the site-selective dual labelling of a protein via chemoselective cysteine modification." Chemical Science 4, no. 9 (2013): 3455. http://dx.doi.org/10.1039/c3sc51333e.
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Gil de Montes, Enrique, Ester Jiménez-Moreno, Bruno L. Oliveira, et al. "Azabicyclic vinyl sulfones for residue-specific dual protein labelling." Chemical Science 10, no. 16 (2019): 4515–22. http://dx.doi.org/10.1039/c9sc00125e.
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Mühlberg, Michaela, Michael G. Hoesl, Christian Kuehne, Jens Dernedde, Nediljko Budisa, and Christian P. R. Hackenberger. "Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds." Beilstein Journal of Organic Chemistry 11 (May 13, 2015): 784–91. http://dx.doi.org/10.3762/bjoc.11.88.
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To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at theN-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding toErythrina cristagallilectin by SPR binding studies.
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Gopalakrishna, R., and W. B. Anderson. "Ca2+- and phospholipid-independent activation of protein kinase C by selective oxidative modification of the regulatory domain." Proceedings of the National Academy of Sciences 86, no. 17 (1989): 6758–62. http://dx.doi.org/10.1073/pnas.86.17.6758.
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The susceptibility of purified protein kinase C to oxidative inactivation by H2O2 was found to be increased by Ca2+ either alone at a high (5 mM) concentration or at a low (approximately 50 microM) concentration along with phosphatidylserine and diacylglycerol and by tumor-promoting phorbol esters even in the absence of Ca2+. This suggested that the membrane-bound and/or catalytically active form of protein kinase C is relatively more susceptible to oxidative inactivation. Although both the regulatory and catalytic domains of protein kinase C were susceptible to oxidative inactivation, a selective modification of the regulatory domain was obtained under mild oxidative conditions by protecting the catalytic site with ATP/Mg2+. Under these conditions there was a loss of both phorbol ester binding and Ca2+/phospholipid-stimulated kinase activity. However, this modified form of enzyme exhibited an increase in Ca2+/phospholipid-independent kinase activity. This suggests that selective oxidative modification of the regulatory domain may negate the requirement for Ca2+ and lipids for activation. Treatment of intact C6 glioma or B16 melanoma cells with H2O2 resulted in a time- and temperature-dependent decrease in Ca2+/phospholipid-dependent protein kinase C activity along with a concomitant transient increase in an oxidatively modified isoform of protein kinase C that exhibited activity in the absence of Ca2+ and phospholipids. Since protein kinase C can initially be activated by mild oxidative modification and subsequently inactivated by further oxidation, this dual activation-inactivation of protein kinase C in response to H2O2 suggests an effective on/off signal mechanism to influence cellular events.
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Li, Na, Jiren Xu, Boheng Liu, Jeevithan Elango, and Wenhui Wu. "Highly Soluble Mussel Foot Protein Enhances Antioxidant Defense and Cytoprotection via PI3K/Akt and Nrf2/HO-1 Pathways." Antioxidants 14, no. 6 (2025): 644. https://doi.org/10.3390/antiox14060644.
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Mussel foot protein is a bioadhesive protein with potential biomedical applications, but its limited solubility and poor biological stability hinder its widespread use. In this study, highly soluble mussel foot protein (HMFP) was successfully extracted using a stepwise selective enzymatic digestion method, with a molecular weight in the range of 11–17 kDa. Furthermore, a dual-functional polyethylene glycol (PEG) derivative of HMFP, designated HMFP-PEG, was synthesized. FTIR analysis confirmed the successful modification of HMFP with PEG, while TGA analysis and SEM observations demonstrated that PEG modification significantly enhanced the stability of the protein. Both HMFP and HMFP-PEG effectively scavenged free radicals, enhanced antioxidant enzyme activity, and reduced MDA levels. Additionally, they activated the PI3K/Akt and Nrf2/HO-1 signaling pathways, inhibiting H2O2-induced cell apoptosis. Notably, HMFP-PEG exhibited superior antioxidant and cell-protective effects compared to HMFP, suggesting that PEG modification improves the functional stability of the protein. This study provides theoretical support for the potential application of HMFP in the biomedical and tissue engineering fields.
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Liu, Haidong, Xiao Li, Yin Shi, Zu Ye, and Xiangdong Cheng. "Protein Tyrosine Phosphatase PRL-3: A Key Player in Cancer Signaling." Biomolecules 14, no. 3 (2024): 342. http://dx.doi.org/10.3390/biom14030342.
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Protein phosphatases are primarily responsible for dephosphorylation modification within signal transduction pathways. Phosphatase of regenerating liver-3 (PRL-3) is a dual-specific phosphatase implicated in cancer pathogenesis. Understanding PRL-3’s intricate functions and developing targeted therapies is crucial for advancing cancer treatment. This review highlights its regulatory mechanisms, expression patterns, and multifaceted roles in cancer progression. PRL-3’s involvement in proliferation, migration, invasion, metastasis, angiogenesis, and drug resistance is discussed. Regulatory mechanisms encompass transcriptional control, alternative splicing, and post-translational modifications. PRL-3 exhibits selective expressions in specific cancer types, making it a potential target for therapy. Despite advances in small molecule inhibitors, further research is needed for clinical application. PRL-3-zumab, a humanized antibody, shows promise in preclinical studies and clinical trials. Our review summarizes the current understanding of the cancer-related cellular function of PRL-3, its prognostic value, and the research progress of therapeutic inhibitors.
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Niwa, Tomoko, Tetsuo Asaki, and Shinya Kimura. "NS-187 (INNO-406), a Bcr-Abl/Lyn Dual Tyrosine Kinase Inhibitor." Analytical Chemistry Insights 2 (January 2007): 117739010700200. http://dx.doi.org/10.4137/117739010700200008.
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Protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl groups of protein side chains, and they play critical roles in regulating cellular signal transduction and other biochemical processes. They are attractive targets for today's drug discovery and development, and many pharmaceutical companies are intensively developing various kinds of protein kinase inhibitors. A good example is the recent success with the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec™) in the treatment of chronic myeloid leukemia. Though imatinib has dramatically improved the treatment of Bcr-Abl-positive chronic myeloid leukemia, resistance is often found in patients with advanced-stage disease. Several mechanisms have been proposed to explain this resistance, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA, increased drug efflux mediated by P-glycoprotein, and activation of the Src-family kinase (SFK) Lyn. We set out to develop a novel drug whose affinity for Abl is higher than that of imatinib and whose specificity in inhibiting Lyn is higher than that of SFK/Abl inhibitors such as dasatinib (Sprycel™) or bosutinib (SKI-606). Our work has led to the development of NS-187 (INNO-406), a novel Abl/Lyn dual tyrosine kinase inhibitor with clinical prospects. To provide an overview of how a selective kinase inhibitor has been developed, this review presents chemical-modification studies carried out with the guidance of molecular modeling, the structural basis for the high potency and selectivity of NS-187 based on the X-ray structure of the NS-187/Abl complex, and the biological profiling of NS-187, including site-directed mutagenesis experiments.
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Adeleye, Samuel A., and Srujana S. Yadavalli. "Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator." PLOS Genetics 20, no. 5 (2024): e1011287. http://dx.doi.org/10.1371/journal.pgen.1011287.
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In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions–Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE’s secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of “one gene, one enzyme, one function”, which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.
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Cheng, Yu-Che, and Sheau-Yann Shieh. "Deubiquitinating enzyme USP3 controls CHK1 chromatin association and activation." Proceedings of the National Academy of Sciences 115, no. 21 (2018): 5546–51. http://dx.doi.org/10.1073/pnas.1719856115.
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Checkpoint kinase 1 (CHK1), a Ser/Thr protein kinase, is modified by the K63-linked ubiquitin chain in response to genotoxic stress, which promotes its nuclear localization, chromatin association, and activation. Interestingly, this bulky modification is linked to a critical residue, K132, at the kinase active site. It is unclear how this modification affects the kinase activity and how it is removed to enable the release of CHK1 from chromatin. Herein, we show that the K63-linked ubiquitin chain at CHK1’s K132 residue has an inhibitory effect on the kinase activity. Furthermore, we demonstrate that this modification can be removed by ubiquitin-specific protease 3 (USP3), a deubiquitinating enzyme that targets K63-linked ubiquitin chains. Wild-type USP3, but not the catalytically defective or nuclear localization sequence-deficient mutants, reduced CHK1 K63-linked ubiquitination. Conversely, USP3 knockdown elevated K63-linked ubiquitination of the kinase, leading to prolonged CHK1 chromatin association and phosphorylation. Paradoxically, by removing the bulky ubiquitin chain at the active site, USP3 also increased the accessibility of CHK1 to its substrates. Thus, our findings on the dual roles of USP3 (namely, one to release CHK1 from the chromatin and the other to open up the active site) provide further insights into the regulation of CHK1 following DNA damage.
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Manahan, Carol L., Madhavi Patnana, Kendall J. Blumer та Maurine E. Linder. "Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae". Molecular Biology of the Cell 11, № 3 (2000): 957–68. http://dx.doi.org/10.1091/mbc.11.3.957.
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To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide–binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein γ subunit (Ste18p) is unusual among Gγsubunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the Gγsubunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of Gβγafter receptor-stimulated release from Gα. The G protein α subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.
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Imam, Hasan, Mohsin Khan, Nandan S. Gokhale, et al. "N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle." Proceedings of the National Academy of Sciences 115, no. 35 (2018): 8829–34. http://dx.doi.org/10.1073/pnas.1808319115.
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N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m6A site in the 5′ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.
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Ma, Qi-Jun, Mei-Hong Sun, Jing Lu, et al. "Phosphorylation of a malate transporter promotes malate excretion and reduces cadmium uptake in apple." Journal of Experimental Botany 71, no. 12 (2020): 3437–49. http://dx.doi.org/10.1093/jxb/eraa121.
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Abstract Heavy metal contamination is a major environmental and human health hazard in many areas of the world. Organic acids sequester heavy metals and protect plant roots from the effects of toxicity; however, it is largely unknown how these acids are regulated in response to heavy metal stress. Here, protein kinase SOS2L1 from apple was functionally characterized. MdSOS2L1 was found to be involved in the regulation of malate excretion, and to inhibit cadmium uptake into roots. Using the DUAL membrane system in a screen of an apple cDNA library with MdSOS2L1 as bait, a malate transporter, MdALMT14, was identified as an interactor. Bimolecular fluorescence complementation, pull-down, and co-immunoprecipitation assays further indicated the interaction of the two proteins. Transgenic analyses showed that MdSOS2L1 is required for cadmium-induced phosphorylation at the Ser358 site of MdALMT14, a modification that enhanced the stability of the MdALMT14 protein. MdSOS2L1 was also shown to enhance cadmium tolerance in an MdALMT14-dependent manner. This study sheds light on the roles of the MdSOS2L1–MdALMT14 complex in physiological responses to cadmium toxicity.
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Firmbach-Kraft, I., and R. Stick. "Analysis of nuclear lamin isoprenylation in Xenopus oocytes: isoprenylation of lamin B3 precedes its uptake into the nucleus." Journal of Cell Biology 129, no. 1 (1995): 17–24. http://dx.doi.org/10.1083/jcb.129.1.17.
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Protein prenylation is a posttranslational modification involving the covalent attachment of a prenyl lipid to a cysteine at or near the COOH terminus of a protein. It is required for membrane localization and efficient function of a number of cytoplasmic as well as nuclear proteins including the proto-oncogenic and activated forms of Ras. Farnesylation in conjunction with a nuclear localization signal has been shown to be necessary to target newly synthesized nuclear lamins to the inner nuclear envelope membrane. It is, however, not clear where in the cell isoprenylation of nuclear lamins takes place. In this study we describe in vivo and in vitro experiments on the isoprenylation of the Xenopus oocyte nuclear lamin B3. We show by kinetic analysis that newly synthesized lamins are isoprenylated in the cytosol of oocytes before uptake into the nucleus. From our data it can be concluded that isoprenylation of lamins in the nucleus, as it is observed under certain conditions of isoprene starvation, represents a default pathway rather than the physiological situation. We further analyzed the capacity of isolated nuclei to carry out isoprenylation of B3. Our results are in line with a dual localization of a protein farnesyltransferase in the cytosol and nuclei of amphibian oocytes. Implications for the possible functions of a nuclear protein farnesyltransferase as well as possible mechanisms of the selective inhibition of farnesylation of cytoplasmic proteins by peptidomimetics are discussed.
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To, Kenneth K. W., Enming Xing, Ross C. Larue, and Pui-Kai Li. "BET Bromodomain Inhibitors: Novel Design Strategies and Therapeutic Applications." Molecules 28, no. 7 (2023): 3043. http://dx.doi.org/10.3390/molecules28073043.
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The mammalian bromodomain and extra-terminal domain (BET) family of proteins consists of four conserved members (Brd2, Brd3, Brd4, and Brdt) that regulate numerous cancer-related and immunity-associated genes. They are epigenetic readers of histone acetylation with broad specificity. BET proteins are linked to cancer progression due to their interaction with numerous cellular proteins including chromatin-modifying factors, transcription factors, and histone modification enzymes. The spectacular growth in the clinical development of small-molecule BET inhibitors underscores the interest and importance of this protein family as an anticancer target. Current approaches targeting BET proteins for cancer therapy rely on acetylation mimics to block the bromodomains from binding chromatin. However, bromodomain-targeted agents are suffering from dose-limiting toxicities because of their effects on other bromodomain-containing proteins. In this review, we provided an updated summary about the evolution of small-molecule BET inhibitors. The design of bivalent BET inhibitors, kinase and BET dual inhibitors, BET protein proteolysis-targeting chimeras (PROTACs), and Brd4-selective inhibitors are discussed. The novel strategy of targeting the unique C-terminal extra-terminal (ET) domain of BET proteins and its therapeutic significance will also be highlighted. Apart from single agent treatment alone, BET inhibitors have also been combined with other chemotherapeutic modalities for cancer treatment demonstrating favorable clinical outcomes. The investigation of specific biomarkers for predicting the efficacy and resistance of BET inhibitors is needed to fully realize their therapeutic potential in the clinical setting.
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Hirschman, Jodi E., та Duane D. Jenness. "Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ". Molecular and Cellular Biology 19, № 11 (1999): 7705–11. http://dx.doi.org/10.1128/mcb.19.11.7705.
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ABSTRACT The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gβγ subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gβγ complex from cells cultured in the presence of [3H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Gγ and Gβγ. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gβγ to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gβγ depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.
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Belousova, Natalya, Galina Mikheeva, Juri Gelovani, and Victor Krasnykh. "Modification of Adenovirus Capsid with a Designed Protein Ligand Yields a Gene Vector Targeted to a Major Molecular Marker of Cancer." Journal of Virology 82, no. 2 (2007): 630–37. http://dx.doi.org/10.1128/jvi.01896-07.
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ABSTRACT The future of genetic interventions in humans critically depends on the selectivity and efficiency of gene transfer to target tissues. The viral gene vectors explored to date cannot selectively transduce the desired targets. While substantial progress has been made in developing targeting strategies for adenovirus (Ad) vectors, future advances in this direction are severely limited by the shortage of naturally existing molecules available for use as targeting ligands. This shortage is due to fundamental and irresolvable differences at the level of both posttranslational modifications and intracellular trafficking between the Ad structural proteins and those natural proteins that are involved in interactions with the cell surface and could otherwise be considered as potential targeting ligands. We hypothesized that this problem could be resolved by altering the natural tropism of Ad vector through incorporation into its capsid of a rationally designed protein ligand, an affibody, whose structural, functional, and biosynthetic properties make it compatible with the Ad assembly process. We tested this hypothesis by redesigning the receptor-binding Ad protein, the fiber, using affibodies specific for human epidermal growth factor receptor type 2 (Her2), a major molecular marker of human tumors. The biosynthesis and folding of these fiber chimeras were fully compatible with Ad virion formation, and the resultant viral vectors were capable of selective delivery of a dual-function transgene to Her2-expressing cancer cells. By establishing the feasibility of this affibody-based approach to Ad vector targeting, the present study lays the foundation for further development of Ad vector technology toward its clinical use.
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Ozols, J., and J. M. Caron. "Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation." Molecular Biology of the Cell 8, no. 4 (1997): 637–45. http://dx.doi.org/10.1091/mbc.8.4.637.
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As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.
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Colombo, Sara, Renato Longhi, Stefano Alcaro, et al. "N-myristoylation determines dual targeting of mammalian NADH-cytochrome b(5) reductase to ER and mitochondrial outer membranes by a mechanism of kinetic partitioning." Journal of Cell Biology 168, no. 5 (2005): 735–45. http://dx.doi.org/10.1083/jcb.200407082.
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Mammalian NADH-cytochrome b(5) reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.
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Williamson, Chad D., and Anamaris M. Colberg-Poley. "Intracellular Sorting Signals for Sequential Trafficking of Human Cytomegalovirus UL37 Proteins to the Endoplasmic Reticulum and Mitochondria." Journal of Virology 84, no. 13 (2010): 6400–6409. http://dx.doi.org/10.1128/jvi.00556-10.
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ABSTRACT Human cytomegalovirus UL37 antiapoptotic proteins, including the predominant UL37 exon 1 protein (pUL37x1), traffic sequentially from the endoplasmic reticulum (ER) through the mitochondrion-associated membrane compartment to the mitochondrial outer membrane (OMM), where they inactivate the proapoptotic activity of Bax. We found that widespread mitochondrial distribution occurs within 1 h of pUL37x1 synthesis. The pUL37x1 mitochondrial targeting signal (MTS) spans its first antiapoptotic domain (residues 5 to 34) and consists of a weak hydrophobicity leader (MTSα) and proximal downstream residues (MTSβ). This MTS arrangement of a hydrophobic leader and downstream proximal basic residues is similar to that of the translocase of the OMM 20, Tom20. We examined whether the UL37 MTS functions analogously to Tom20 leader. Surprisingly, lowered hydropathy of the UL37x1 MTSα, predicted to block ER translocation, still allowed dual targeting of mutant to the ER and OMM. However, increased hydropathy of the MTS leader caused exclusion of the UL37x1 high-hydropathy mutant from mitochondrial import. Conversely, UL37 MTSα replacement with the Tom20 leader did not retarget pUL37x1 exclusively to the OMM; rather, the UL37x1-Tom20 chimera retained dual trafficking. Moreover, replacement of the UL37 MTSβ basic residues did not reduce OMM import. Ablation of the MTSα posttranslational modification site or of the downstream MTS proline-rich domain (PRD) increased mitochondrial import. Our results suggest that pUL37x1 sequential ER to mitochondrial trafficking requires a weakly hydrophobic leader and is regulated by MTSβ sequences. Thus, HCMV pUL37x1 uses a mitochondrial importation pathway that is genetically distinguishable from that of known OMM proteins.
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Shimizu, Tatsuhiro, Takafumi Nomachi, Kunihiro Matsumoto, and Naoki Hisamoto. "A cytidine deaminase regulates axon regeneration by modulating the functions of the Caenorhabditis elegans HGF/plasminogen family protein SVH-1." PLOS Genetics 20, no. 7 (2024): e1011367. http://dx.doi.org/10.1371/journal.pgen.1011367.
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The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp–His–Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA.
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Malanchuk, Oksana, Anna Bdzhola, Sergii Palchevskyi, et al. "Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation." International Journal of Molecular Sciences 25, no. 16 (2024): 8747. http://dx.doi.org/10.3390/ijms25168747.
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Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a “dual anchor” mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.
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Lee, Choong-Eun, Tam Tran, Seol-Hee Kim, Ki-Sa Sung, and Cheol-Yong Choi. "Regulation of IL-4-induced STAT6 activation by SUMOylation (P6307)." Journal of Immunology 190, no. 1_Supplement (2013): 184.15. http://dx.doi.org/10.4049/jimmunol.190.supp.184.15.
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Abstract As a key signaling molecule in the cytokine-mediated transcriptional activation STATs are shown to undergo PTMs including phosphorylation, acetylation, methylation, ubiquitination and SUMOylation. The SUMO-modification of STAT1 has been implicated in the regulation of protein stability, nuclear translocation, and transcriptional activation, probably through the reduction of the formation and life-span of the active dimer. As a part of regulation of IL-4 signaling mechanism, we have investigated the role of SUMOylation on STAT6 activation. We have identified potential SUMOylation sites on STAT6 by SUMOplot prediction program analysis and observed the IL-4-induced SUMOlyation of STAT6 in HeLa cells. The introduction of mutation at a potential SUMO site K636 and transfection of shRNA of SUMO-conjugating enzyme UBC9 resulted in the inhibition of tyrosine phosphorylation at Y641 induced by IL-4. The results indicate that endogenous SUMOylation reaction of STAT6 is critical for IL-4-induced STAT6 phosphorylation. Conversely, the forced expression of SUMO-1 and STAT6 caused cytosolic relocalization of both STAT6 and phospho-STAT6 induced by IL-4 as demonstrated by Western blot and confocal analysis. Our data suggest that SUMO-modification of STAT6 may play dual roles in the regulation of phosphorylation and nuclear translocation of STAT6 during IL-4 signal transduction.
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EVANS, Paul C., Huib OVAA, Maureen HAMON, et al. "Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity." Biochemical Journal 378, no. 3 (2004): 727–34. http://dx.doi.org/10.1042/bj20031377.
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Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which ‘trim’ polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-κB (nuclear factor κB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IκBα (inhibitor of κB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKγ (IκB kinase γ)/NEMO (NF-κB essential modifier) signal adaptors relies on their modification with ‘nonclassical’ forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-κB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.
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Mebatsion, Teshome, Stefan Verstegen, Leonarda T. C. De Vaan, Angela Römer-Oberdörfer, and Carla C. Schrier. "A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos." Journal of Virology 75, no. 1 (2001): 420–28. http://dx.doi.org/10.1128/jvi.75.1.420-428.2001.
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ABSTRACT Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3′-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.
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Gantz, Valentino M., Nijole Jasinskiene, Olga Tatarenkova, et al. "Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi." Proceedings of the National Academy of Sciences 112, no. 49 (2015): E6736—E6743. http://dx.doi.org/10.1073/pnas.1521077112.
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Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.
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Zimmer, Markus, Siegfried Scherer та Martin J. Loessner. "Genomic Analysis of Clostridium perfringens Bacteriophage φ3626, Which Integrates into guaA and Possibly Affects Sporulation". Journal of Bacteriology 184, № 16 (2002): 4359–68. http://dx.doi.org/10.1128/jb.184.16.4359-4368.2002.
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ABSTRACT Two temperate viruses, φ3626 and φ8533, have been isolated from lysogenic Clostridium perfringens strains. Phage φ3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3′-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of φ3626 with onset of sporulation in C. perfringens. We found that the φ3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3′ end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.
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Pattabiraman, Vijaya R., Matilde Arévalo Ruiz, Régis Boehringer, et al. "Abstract 2138: Creating next-generation biologics using a novel chemistry platform technology." Cancer Research 82, no. 12_Supplement (2022): 2138. http://dx.doi.org/10.1158/1538-7445.am2022-2138.
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Abstract Bright Peak Therapeutics is developing a portfolio of differentiated biotherapeutics using chemistry for applications in immuno-oncology and autoimmune diseases. Our unique chemical protein synthesis and engineering platform allows us to fine-tune cytokines and other proteins to interrogate and modulate biological functions by incorporating new functional modifications. Standard recombinant bacterial or cellular expression systems used to produce proteins are largely restricted to using canonical amino acids, which limits access to diverse modifications that can bestow additional functional properties. With chemical protein synthesis technology, canonical and non-canonical modifications including conjugation handles can be easily introduced, ultimately enabling a medicinal chemistry approach for engineering cytokine structures. Enhanced cytokines with differentiated biology developed using this approach can be further elaborated by conjugating to a diverse array of molecules. We first applied our technology platform to identify BPT-143, a rationally designed enhanced IL-2 variant currently in IND-enabling studies. BPT-143 is engineered to have enhanced binding to IL2Rβ and no binding to IL2Rα for improved efficacy and safety independent of the conjugation to a half-life extending 30 kDa PEG. The chemical synthesis technology is robust, reproducible, and scalable. We are applying our platform to enhance a number of other cytokines for use in immuno-oncology and autoimmune diseases. Additionally, our synthetically engineered cytokines can be easily conjugated to monoclonal antibodies as ‘payloads’ using a distinct chemical conjugation technology. A rapid and simple chemical process allows site-selective conjugation of our engineered cytokines to existing antibodies ‘as-is’ to generate novel immunocytokines (IC). This ‘off-the-shelf’ approach is orthogonal to recombinant fusion methods to create ICs and does not require complex recombinant protein expression optimization and lengthy cell-line development. Moreover, it allows rapid screening of cytokine payloads in a structure-activity relationship (SAR) format to identify dual-targeting ICs with precisely tailored properties to generate the desired biological effect. We have prepared a number of ICs including anti-PD-1/IL-2 ICs with various drug-antibody ratio (DAR) and conjugation sites within the antibody. We will provide an overview of the platform technology and present highlights of its application for discovery and development of designer therapeutic cytokines and ICs. Citation Format: Vijaya R. Pattabiraman, Matilde Arévalo Ruiz, Régis Boehringer, Benoit Hornsperger, Roy Meoded, Robert C. Tam, Bertolt Kreft. Creating next-generation biologics using a novel chemistry platform technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2138.
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Albitar, Obaida, Crystal M. D’Souza, and Ernest A. Adeghate. "Effects of Lipoproteins on Metabolic Health." Nutrients 16, no. 13 (2024): 2156. http://dx.doi.org/10.3390/nu16132156.
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Lipids are primarily transported in the bloodstream by lipoproteins, which are macromolecules of lipids and conjugated proteins also known as apolipoproteins. The processes of lipoprotein assembly, secretion, transportation, modification, and clearance are crucial components of maintaining a healthy lipid metabolism. Disruption in any of these steps results in pathophysiological abnormalities such as dyslipidemia, obesity, insulin resistance, inflammation, atherosclerosis, peripheral artery disease, and cardiovascular diseases. By studying these genetic mutations, researchers can gain valuable insights into the underlying mechanisms that govern the relationship between protein structure and its physiological role. These lipoproteins, including HDL, LDL, lipoprotein(a), and VLDL, mainly serve the purpose of transporting lipids between tissues and organs. However, studies have provided evidence that apo(a) also possesses protective properties against pathogens. In the future, the field of study will be significantly influenced by the integration of recombinant DNA technology and human site-specific mutagenesis for treating hereditary disorders. Several medications are available for the treatment of dyslipoproteinemia. These include statins, fibrates, ezetimibe, niacin, PCSK9 inhibitors, evinacumab, DPP 4 inhibitors, glucagon-like peptide-1 receptor agonists GLP1RAs, GLP-1, and GIP dual receptor agonists, in addition to SGLT2 inhibitors. This current review article exhibits, for the first time, a comprehensive reflection of the available body of publications concerning the impact of lipoproteins on metabolic well-being across various pathological states.
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Deng, Ou, Xueli Li, Vinayak C. Palve, Bin Fang, Damon R. Reed, and Uwe Rix. "Abstract C054: PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition." Molecular Cancer Therapeutics 22, no. 12_Supplement (2023): C054. http://dx.doi.org/10.1158/1535-7163.targ-23-c054.
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Abstract BACKGROUND: Ewing’s sarcoma (EWS) is a highly aggressive bone and soft tissue tumor with poor prognosis and an urgent need for novel therapies. We have identified that silencing of mono-ADP-ribosyltransferase PARP16 enhanced sensitivity of EWS cell lines to PARP1 inhibition. In this study, we explored the molecular mechanism of EWS vulnerability towards PARP16 targeting, alone and in conjunction with PARP1 inhibition, which may lead to development of PARP16-selective or dual targeting PARP1/16 inhibitors as new drugs for EWS. METHODS: The effect of PARP16 on cell viability was analyzed using RNA interference with various siRNAs and doxycycline-inducible PARP16 shRNAs, as well as CRISPR/Cas9-mediated genomic deletion. The changes on total protein expression upon silencing of PARP16 with or without PARP1 inhibition was determined by mass spectrometry (MS). Protein interaction partners and downstream effectors of PARP16 were determined by Co-IP/MS and ADP-ribosylation proteomics, respectively. RESULTS: Transient knockdown of PARP16 significantly reduced cell survival in EWS cell lines, particularly in combination with olaparib. Quantitative protein expression analysis by MS suggested doxycycline-induced shRNA-mediated PARP16 knockdown significantly reduced MYC protein expression in addition to PARP16 itself. Depletion of PARP16 in Cas9-expressing SK-N-MC EWS cells using different lentiviral sgRNAs further confirmed that targeting of PARP16 reduces cell survival and MYC expression, particularly in combination with PARP1 inhibition. Co-IP/MS using GFP-Trap agarose beads in mGFP/mGFP-PARP16 expressing cells suggested interaction of PARP16 with proteins that regulate post-translational modification of MYC in EWS cells. Further validation is ongoing. CONCLUSION: Our data suggest that targeting PARP16 confers vulnerability to EWS cells either alone or in conjunction with PARP1 inhibition and that it is involved in regulation of MYC. Identification of novel PARP16 substrates and interaction partners is expected to produce molecular markers that enable the development of novel PARP16 inhibitors. Citation Format: Ou Deng, Xueli Li, Vinayak C Palve, Bin Fang, Damon R Reed, Uwe Rix. PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C054.
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Nakamura, Fumihiko, Laiqiang Huang, Kersi Pestonjamasp, Elizabeth J. Luna, and Heinz Furthmayr. "Regulation of F-Actin Binding to Platelet Moesin In Vitro by Both Phosphorylation of Threonine 558 and Polyphosphatidylinositides." Molecular Biology of the Cell 10, no. 8 (1999): 2669–85. http://dx.doi.org/10.1091/mbc.10.8.2669.
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Activation of human platelets with thrombin transiently increases phosphorylation at558threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with α- or β/γ-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, ∼1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin’s high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.
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Evans, J. L., B. Quistorff, and L. A. Witters. "Hepatic zonation of acetyl-CoA carboxylase activity." Biochemical Journal 270, no. 3 (1990): 665–72. http://dx.doi.org/10.1042/bj2700665.
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The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5′-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.
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Hoyt, Emily A., Pedro M. S. D. Cal, Bruno L. Oliveira, and Gonçalo J. L. Bernardes. "Contemporary approaches to site-selective protein modification." Nature Reviews Chemistry 3, no. 3 (2019): 147–71. http://dx.doi.org/10.1038/s41570-019-0079-1.
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Lin, Yuya A., Justin M. Chalker, and Benjamin G. Davis. "Olefin Metathesis for Site-Selective Protein Modification." ChemBioChem 10, no. 6 (2009): 959–69. http://dx.doi.org/10.1002/cbic.200900002.
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Gordon, Britten, Elizabeth Muhowski, Janani Ravikrishnan, et al. "Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168." Blood 142, Supplement 1 (2023): 416. http://dx.doi.org/10.1182/blood-2023-178259.
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Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p&lt;0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p&lt;0.001, n=10 and 51%, p&lt;0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p&lt;0.001, n=8; CCL4: 93%, p&lt;0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p&lt;0.0001, n=3; T474I: 99%, p&lt;0.0001, n=3) and CCL4 (C481S: 97%, p&lt;0.0001, n=3; T474I: 98%, p&lt;0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p&lt;0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).
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Brewster, Richard C., and Alison N. Hulme. "Halomethyl-Triazoles for Rapid, Site-Selective Protein Modification." Molecules 26, no. 18 (2021): 5461. http://dx.doi.org/10.3390/molecules26185461.
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Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.
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Hamdy, Rania, Bahgat Fayed, Ahmed Mostafa, et al. "Iterated Virtual Screening-Assisted Antiviral and Enzyme Inhibition Assays Reveal the Discovery of Novel Promising Anti-SARS-CoV-2 with Dual Activity." International Journal of Molecular Sciences 22, no. 16 (2021): 9057. http://dx.doi.org/10.3390/ijms22169057.
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Unfortunately, COVID-19 is still a threat to humankind and has a dramatic impact on human health, social life, the world economy, and food security. With the limited number of suggested therapies under clinical trials, the discovery of novel therapeutic agents is essential. Here, a previously identified anti-SARS-CoV-2 compound named Compound 13 (1,2,5-Oxadiazole-3-carboximidic acid, 4,4′-(methylenediimino) bis,bis[[(2-hydroxyphenyl)methylene]hydrazide) was subjected to an iterated virtual screening against SARS-CoV-2 Mpro using a combination of Ligand Designer and PathFinder. PathFinder, a computational reaction enumeration tool, was used for the rapid generation of enumerated structures via default reaction library. Ligand designer was employed for the computerized lead optimization and selection of the best structural modification that resulted in a favorable ligand–protein complex. The obtained compounds that showed the best binding to Mpro were re-screened against TMPRSS2, leading to the identification of 20 shared compounds. The compounds were further visually inspected, which resulted in the identification of five shared compounds M1–5 with dual binding affinity. In vitro evaluation and enzyme inhibition assay indicated that M3, an analogue of Compound 13 afforded by replacing the phenolic moiety with pyridinyl, possesses an improved antiviral activity and safety. M3 displayed in vitro antiviral activity with IC50 0.016 µM and Mpro inhibition activity with IC50 0.013 µM, 7-fold more potent than the parent Compound 13 and potent than the antivirals drugs that are currently under clinical trials. Moreover, M3 showed potent activity against human TMPRSS2 and furin enzymes with IC50 0.05, and 0.08 µM, respectively. Molecular docking, WaterMap analysis, molecular dynamics simulation, and R-group analysis confirmed the superiority of the binding fit to M3 with the target enzymes. WaterMap analysis calculated the thermodynamic properties of the hydration site in the binding pocket that significantly affects the biological activity. Loading M3 on zinc oxide nanoparticles (ZnO NPs) increased the antiviral activity of the compound 1.5-fold, while maintaining a higher safety profile. In conclusion, lead optimized discovery following an iterated virtual screening in association with molecular docking and biological evaluation revealed a novel compound named M3 with promising dual activity against SARS-CoV-2. The compound deserves further investigation for potential clinical-based studies.
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Keijzer, Jordi F., Judith Firet, and Bauke Albada. "Site-selective and inducible acylation of thrombin using aptamer-catalyst conjugates." Chemical Communications 57, no. 96 (2021): 12960–63. http://dx.doi.org/10.1039/d1cc05446e.
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Functionalizing a protein-binding aptamer with an acylation catalyst leads to site-selective modification of the target protein in proximity to the aptamer–protein interface. This protein modification can be switched ON or OFF by an external trigger.
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Rosen, Christian B., and Matthew B. Francis. "Targeting the N terminus for site-selective protein modification." Nature Chemical Biology 13, no. 7 (2017): 697–705. http://dx.doi.org/10.1038/nchembio.2416.
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ANTOS, J., and M. FRANCIS. "Transition metal catalyzed methods for site-selective protein modification." Current Opinion in Chemical Biology 10, no. 3 (2006): 253–62. http://dx.doi.org/10.1016/j.cbpa.2006.04.009.
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Cai, Mengyuan, Peng Yin, Ziwen Wang, et al. "Abstract 4401: Dual regulation of frizzled receptors (FZD1/7) by IGF2BP3: A novel oncogenic event promotes stem-like properties and reduces carboplatin chemosensitivity in Triple-negative breast cancer." Cancer Research 85, no. 8_Supplement_1 (2025): 4401. https://doi.org/10.1158/1538-7445.am2025-4401.
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Abstract Background: Triple-negative breast cancer (TNBC) is primarily driven by cancer stem cells (CSCs), resulting in treatment resistance after chemotherapy. Emerging evidence suggests that TNBC-CSC transformation is strongly dependent on m6A modification. We aim to characterize the specific m6A regulatory factor in TNBC-CSCs and its underlying mechanism. Methods: Transcriptome-based screening was used to determine the main m6A regulator in TNBC-CSCs. Mammosphere formation assay and flow cytometry were used to evaluate the role of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in regulating stemness in TNBC in vitro. Limiting dilution assay was used to assess the CSC frequency in vivo. Multi-omics data, including RNA immunoprecipitation sequencing (RIP-seq), Methylated RIP sequencing ((MeRIP-seq) and RNA sequencing data, were integrated to investigate the mRNA targets of IGF2BP3. RNA probes and IGF2BP3 truncations were generated to detect the direct binding location of IGF2BP3 and its target FZD1/7 mRNAs. Comet assay and homologous recomination (HR) assay were used to assess the effect of IGF2BP3 knockdown and FZD1/7 inhibitor, Fz7-21 in combination with carboplatin (CBP). Results: We demonstrated that IGF2BP3 was the dominant m6A regulator in TNBC-CSCs. Its knockdown increased chemosensitivity and reduced tumor recurrence rate after CBP therapy in vivo. Furthermore, IGF2BP3-m6A-FZD1/7 axis might act as a pivotal oncogenic event in TNBC. Stem-like properties, including higher homologous recombination repair (HRR) efficacy and chemoresistance to CBP driven by frizzled receptors 1/7 (FZD1/7), were pharmacologically targeted by a small-molecule inhibitor, Fz7-21. Mechanistic studies unveiled that the RNA-recognition motif (RRM) domain of IGF2BP3 was the functional site to recognize m6A modification on the 3'-terminal of FZD1/7 mRNAs, leading to increased RNA stability. Enhanced stability elevated the expression levels of these two transcripts, leading to FZD1/7 heterodimerization and the activation of β-catenin pathway. This was marked by the nuclear translocation of non-phosphorylated (Ser37 and Thr41) β-catenin. Conclusions: Our study highlights the critical oncogenic role of IGF2BP3 in promoting stem-like properties and reduces the TNBC sensitivity to CBP treatment. Targeting IGF2BP3 and FZD1/7 with inhibitors has the therapeutic potential to disrupt cancer stemness and reduce clinical drug dosage of CBP in TNBC patients. Citation Format: Mengyuan Cai, Peng Yin, Ziwen Wang, Yuzhou Huang, Yuxin Wang, Kefei Wu, Xu Zhang, Liang Shi, Jifu Wei, Qiang Ding. Dual regulation of frizzled receptors (FZD1/7) by IGF2BP3: A novel oncogenic event promotes stem-like properties and reduces carboplatin chemosensitivity in Triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4401.
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Moody, Paul, Vijay Chudasama, Ramiz I. Nathani, et al. "A rapid, site-selective and efficient route to the dual modification of DARPins." Chem. Commun. 50, no. 38 (2014): 4898–900. http://dx.doi.org/10.1039/c4cc00053f.
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Herein we describe a rapid, simple method for dual modification of DARPins by introduction of cysteine mutations at specific positions that results in a vast difference in their thiol nucleophilicity, allowing for sequential modification.
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Scumaci, Domenica, Erika Olivo, Claudia Vincenza Fiumara, et al. "DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation." Cells 9, no. 9 (2020): 1968. http://dx.doi.org/10.3390/cells9091968.
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Enhanced glycolysis is a hallmark of breast cancer. In cancer cells, the high glycolytic flux induces carbonyl stress, a damaging condition in which the increase of reactive carbonyl species makes DNA, proteins, and lipids more susceptible to glycation. Together with glucose, methylglyoxal (MGO), a byproduct of glycolysis, is considered the main glycating agent. MGO is highly diffusible, enters the nucleus, and can react with easily accessible lysine- and arginine-rich tails of histones. Glycation adducts on histones undergo oxidization and further rearrange to form stable species known as advanced glycation end-products (AGEs). This modification alters nucleosomes stability and chromatin architecture deconstructing the histone code. Formation of AGEs has been associated with cancer, diabetes, and several age-related diseases. Recently, DJ-1, a cancer-associated protein that protects cells from oxidative stress, has been described as a deglycase enzyme. Although its role in cell survival results still controversial, in several human tumors, its expression, localization, oxidation, and phosphorylation were found altered. This work aimed to explore the molecular mechanism that triggers the peculiar cellular compartmentalization and the specific post-translational modifications (PTM) that, occurring in breast cancer cells, influences the DJ-1 dual role. Using a proteomic approach, we identified on DJ-1 a novel threonine phosphorylation (T125) that was found, by the in-silico tool scansite 4, as part of a putative Akt consensus. Notably, this threonine is in addition to histidine 126, a key residue involved in the formation of catalytic triade (glu18-Cys106-His126) inside the glioxalase active site of DJ. Interestingly, we found that pharmacological modulation of Akt pathway induces a functional tuning of DJ-1 proteoforms, as well as their shuttle from cytosol to nucleus, pointing out that pathway as critical in the development of DJ-1 pro-tumorigenic abilities. Deglycase activity of DJ-1 on histones proteins, investigated by coupling 2D tau gel with LC-MS/MS and 2D-TAU (Triton-Acid-Urea)-Western blot, was found correlated with its phosphorylation status that, in turn, depends from Akt activation. In normal conditions, DJ-1 acts as a redox-sensitive chaperone and as an oxidative stress sensor. In cancer cells, glycolytic rewiring, inducing increased reactive oxygen species (ROS) levels, enhances AGEs products. Alongside, the moderate increase of ROS enhances Akt signaling that induces DJ-1-phosphorylation. When phosphorylated DJ-1 increases its glyoxalase activity, the level of AGEs on histones decreases. Therefore, phospho-DJ-1 prevents glycation-induced histones misregulation and its Akt-related hyperactivity represents a way to preserve the epigenome landscape sustaining proliferation of cancer cells. Together, these results shed light on an interesting mechanism that cancer cells might execute to escape the metabolic induced epigenetic misregulation that otherwise could impair their malignant proliferative potential.
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Abbas, Sk Jahir, Sabina Yesmin, Sandeepa K. Vittala, et al. "Target Bioconjugation of Protein Through Chemical, Molecular Dynamics, and Artificial Intelligence Approaches." Metabolites 14, no. 12 (2024): 668. https://doi.org/10.3390/metabo14120668.
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Covalent modification of proteins at specific, predetermined sites is essential for advancing biological and biopharmaceutical applications. Site-selective labeling techniques for protein modification allow us to effectively track biological function, intracellular dynamics, and localization. Despite numerous reports on modifying target proteins with functional chemical probes, unique organic reactions that achieve site-selective integration without compromising native functional properties remain a significant challenge. In this review, we delve into site-selective protein modification using synthetic probes, highlighting both chemical and computational methodologies for chemo- and regioselective modifications of naturally occurring amino acids, as well as proximity-driven protein-selective chemical modifications. We also underline recent traceless affinity labeling strategies that involve exchange/cleavage reactions and catalyst tethering modifications. The rapid development of computational infrastructure and methods has made the bioconjugation of proteins more accessible, enabling precise predictions of structural changes due to protein modifications. Hence, we discuss bioconjugational computational approaches, including molecular dynamics and artificial intelligence, underscoring their potential applications in enhancing our understanding of cellular biology and addressing current challenges in the field.
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Chalker, Justin M., Gonçalo J. L. Bernardes, and Benjamin G. Davis. "A “Tag-and-Modify” Approach to Site-Selective Protein Modification." Accounts of Chemical Research 44, no. 9 (2011): 730–41. http://dx.doi.org/10.1021/ar200056q.
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Shadish, Jared A., and Cole A. DeForest. "Site-Selective Protein Modification: From Functionalized Proteins to Functional Biomaterials." Matter 2, no. 1 (2020): 50–77. http://dx.doi.org/10.1016/j.matt.2019.11.011.
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ElSohly, Adel M., and Matthew B. Francis. "Development of Oxidative Coupling Strategies for Site-Selective Protein Modification." Accounts of Chemical Research 48, no. 7 (2015): 1971–78. http://dx.doi.org/10.1021/acs.accounts.5b00139.
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