Academic literature on the topic 'Site-selective protein dual modification'

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_i 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.

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.

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.

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.

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.

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.

Site-selective protein modification has become an important tool to study protein functions in chemical biology. In the preliminary work, allyl sulfides were found to be reactive substrates in aqueous cross-metathesis (CM) enabling the first examples of protein modification via this approach. In order to access the enhanced CM reactivity of allyl sulfide on proteins, facile chemical methods to install S-allyl cysteine on protein surface were developed. In particular, a cysteine-specific allylating reagent – allyl selenocyanate was used on protein substrate for the first time. The substrate scope of allyl sulfide-tagged proteins and factors that affect the outcome of CM was also investigated. A range of metathesis substrates containing different olefin tether of various lengths were screened; allyl ethers were found to be most suitable as CM partners. By reducing the steric hindrance around the allyl sulfide on protein surface through a chemical spacer, the rate and conversion of metathesis reaction on proteins was greatly enhanced. Moreover, allyl selenides were found to be more reactive than allyl sulfides in CM and enabled reactions with substrates that were previously impossible for the corresponding sulfur-analogue. Through this work, substrate selection guidelines for successful metathesis reaction on proteins were established. Rapid Se-relayed CM was further investigated through biomimetic chemical access to Se-allyl selenocysteine (Seac) via dehydroalanine. On-protein reaction kinetics revealed rate constants of Seac-mediated CM to be comparable or superior to off-protein rates of many current bioconjugations. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (K9Ac, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. A Cope-type selenoxide elimination subsequently allowed the removal of such modification to regenerate dehydroalanine. Finally, preliminary research efforts towards metabolic incorporation of allyl sulfide-containing amino acid into proteins, and CM on cell surfaces were discussed.

Disulfide bonds represent an important target for site-selective protein modification, particularly via the strategy of functional re-bridging. Reduction of interchain disulfide bonds, followed by their re-bridging allows proteins to be functionalised in a site-selective manner whilst retaining the stability and integrity offered by the original bridge. This work describes the design and development of two distinct pyridazinedione-based technologies that, through the conduit of functional disulfide re-bridging, enable the synthesis of antibody – drug conjugates with hitherto unmet levels of control and homogeneity. As proteins often contain multiple disulfide bonds that are critical to conformation and stability, reagents that allow functional disulfide re-bridging without disulfide scrambling (non-native disulfide re-bridging) in multiple disulfide containing systems are critical for the success of this method. The first presented technology is a molecule that is capable of both reducing and re-bridging disulfide bonds, enabling a rapid and efficient one-reagent protocol for the functionalisation of disulfide containing proteins, moreover, it does so in such a way that native disulfide configuration is retained via a high local concentration effect. This novel pyridazinedione scaffold has been shown to functionalise a variety of therapeutically relevant proteins, including the widely used mAb HerceptinTM, enabling the synthesis of homogenous antibody – drug conjugates from a native mAb. Shifting focus from homogeneity to control over drug loading, the second presented technology is a single pyridazinedione-based molecule that contains four cysteine reactive centres and only one bioorthogonal reactive handle, which enables the generation of antibody conjugates with a loading of two modules. A loading of two is desirable for many reasons, especially in the context of large, hydrophobic payloads, which are increasingly popular for use in antibody-drug conjugates. A loading of two drugs per antibody has been shown to provide an optimal balance between efficacy and biophysical properties in many cases. A reliable method based on a native antibody scaffold without the use of enzymes or harsh oxidative conditions has hitherto not been achieved. The use of native antibodies has several advantages in terms of cost, practicality, accessibility and time. Thus, a novel, reliable method of furnishing antibody conjugates with a loading of two modules starting from a native antibody scaffold was developed.

Antibody–drug conjugates (ADCs) are a particularly promising class of antibody-based therapeutics. They are commonly referred to as “magic-bullet” therapy due to the ability to seek and destroy primarily diseased cells within the body (e.g. cancer cells). Critical to this strategy is the availability of effective methodologies to link an antibody to other molecules (e.g. cytotoxic drugs, prodrugs). Although current approaches offer great promise for the development of ADC constructs, they often are not selective and yield heterogeneous product mixtures. Other strategies require mutations with natural or unnatural amino acids, which are generally associated with low expression yields and post-translational issues, whilst some result in the loss of vital disulfide bonds. Furthermore, most of the current peptide/payload linker technologies have the potential to attach only one moiety, greatly limiting their scope and flexibility. To tackle these issues, a novel class of tuneable reagents based on pyridazinedione (PD) moieties was developed. These reagents are readily accessible from inexpensive starting materials in 3 – 4 scalable steps and overcome the problems stated above. Indeed, the PD-based reagents have exquisite selectivity towards cysteines over lysines, even at high temperature and/or with an excess of reagent. When reacted with reduced native disulfide bonds of antibodies or antibody fragments, PDs rapidly and quantitatively form a stable and rigid 2-carbon bridge between the two cysteines side-chains without affecting the structure and biological activity of antibodies. The resulting conjugates have also been demonstrated to have exceptional resistance to hydrolysis at various extreme pHs and temperatures over an extended period of time. Finally, the PD-conjugates possess two orthogonal handles that were readily modified with two different moieties to generate highly functionalised homogeneous constructs with application not only in antibody therapeutics but also in imaging and diagnostics.

The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.

Carbohydrates and proteins represent two large groups of biomolecules which are tremendously important for biological processes in health and disease state. Although protein-structures are encoded in the genome, cellular glycan structures are template independent and can only be addressed in an indirect manner. The development of metabolic oligosaccharide engineering (MOE) gave rise to new methods to study carbohydrate structures in the context of different disease settings and in different organisms. While in many cases mannose derivatives are used to study the sialic acid structures in cancer cells, this work presents the results on the metabolic incorporation of galactose derivatives into cell membrane glycans of human hepatic cells. Three unnatural galactose derivatives containing terminal alkene groups in C2 or C6 position were synthesized and their reaction rates in inverse electron demand Diels Alder reactions (iEDDA) were evaluated, by using a high-throughput screening method in 96-well plates. It was shown that none of the developed galactose derivatives exhibit any cell toxic effect in HepG2 or Huh7 cell lines. Furthermore, all monosaccharides could be successfully incorporated in cell membrane glycan structures of both cell lines and the localization on the cell membrane was confirmed by co-localization with a plasma membrane dye. After developing this incorporation and labeling strategy of unnatural galactose derivatives in the cell membrane of human hepatic cells, the change in incorporation during an infection of these cells with Plasmodium berghei sporozoites was investigated. By using different techniques, such as confocal microscopy, flow cytometry and imaging flow cytometry, only a small trend for an increased uptake of the unnatural galactose derivative in P. berghei infected cells was observed. To explain this result, the pathway for the diffusion of the unnatural galactose derivative was determined. The application of specific and non-specific inhibitors for the glucose transporter GLUT1 revealed that this transporter is involved in the delivery of galactose derivatives into cultured cells. The enhanced translocation of this transporter to the surface of infected hepatic cells explains the observed tendency for an increased incorporation of the unnatural galactose derivative in these cells. Apart from cell studies, MOE was applied for the first time to study a possible transfer of galactose monosaccharides from the mosquito host to the parasite. Biosynthetic pathways for glycan assembly in the parasite are poorly understood. Suggestions on the participation of the mosquito host in some of these pathways, led to the idea to apply MOE in this situation. It was possible to show an uptake of the presented galactose derivatives by the mosquito but only reduced transfer to the parasite seems to occur. In addition to the development of monofunctional galactose derivatives, also a bifunctional derivative containing two orthogonal reporter groups was synthesized. However, so far it was not possible to achieve a metabolic incorporation or labeling of this derivative on cell membrane glycans. After developing cellular tools to study carbohydrate structures, a site-selective method for protein modification was generated, to be used for the development of new glycoconjugate vaccine candidates. By introducing selectively two dehydroalanine residues in place of the disulfide bond C186-C201 of the immunogenic protein CRM197, a new chemical moiety for the conjugation of carbohydrate antigens was obtained. It was shown that these moieties can be used for the selective introduction of polysaccharide antigens from group B Streptococcus (GBS) or Streptococcus pneumoniae. Both types of glycoconjugates could be synthesized and first trials on the purification methods were undertaken. This concept will be developed further for future vaccine candidates. Finally, a synthetic method was developed which could facilitate the synthesis of defined antigenic oligosaccharide structures. This method uses the thiophilic promoter O-mesitylenesulfonylhydroxylamine (MSH) for the activation of thioglycoside donors. It was demonstrated that different thioglycoside donors are activated with different kinetics, depending on the presented protecting groups or the anomeric leaving group. Apart from applying the developed activation method for the synthesis of several glycosylation products, the sequential activation of S-alkyl before S-phenyl anomeric groups was shown during the synthesis of a model trisaccharide. Overall, bio-orthogonal methods were developed and applied for the investigation of carbohydrate structures in the context of malaria disease, and for the site-selective modification of protein carriers during the development of glycoconjugate vaccine candidates.

Alzheimer's disease (AD) is characterized by selective loss of neurons in the hippocampus and neocortex due to abnormalities in proteins, mainly Aβ peptide and tau protein, in the form of abnormal protein aggregations or depositions in neurons. Recently oxidative/nitrosative stress has been identified as an important facilitator of neurodegeneration in AD. Cysteine-dependent proteins are known to be associated with the neurodegenerative process. Such cysteine-dependent enzyme proteins are proteases, antioxidant enzymes, kinases, phosphatases, and also non-enzymatic proteins such that utilize cysteine as a structural part of the catalytic site. This chapter deals with the role of cysteine in handling reactive oxygen/nitrogen species during oxidative/nitrosative stress and posttranslational modification of proteins causing protein misfolding or protein aggregation during neurodegeneration associated with AD.

Abstract Posttranslational modification of proteins allows an expansion of the side-chain chemistry of the translationally encoded amino acids to widen the range of protein activities. The ability to reversibly modify proteins at specific times provides a flexible means for regulating protein function on a wide range of time scales. A selective list of post- translational modifications is provided in Table 129–1. To create this broad repertoire, a large number of protein-modifying enzymes and cofactors evolved to catalyze these additions, typically on specific consensus sites within target proteins. For a given posttranslational mechanism to function, both the trans-acting modifying enzymes and the cisacting sites of modification on protein targets must be intact. A wide range of human genetic diseases have already been found to involve deficiencies in both the modifying enzymes and their recognition sites on protein targets, and more examples are likely on the way. Among the most highly studied posttranslational mechanisms is the addition of the 76 amino acid protein ubiquitin to other proteins, a modification used (1) to tag proteins for proteolytic destruction by a pathway to be described here and (2) to more directly modify the activity of proteins, in many cases through the assembly of protein complexes on site of monoubiquitination or on assembled ubiquitin chains.