Literatura académica sobre el tema "SAnk1.5"

Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5).Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk–associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site.

The sarcoplasmic reticulum (SR) serves as the Ca2+ reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γcyto-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γcyto-actin and Tmod3 defined an SR microdomain that was distinct from another Z line–flanking SR microdomain containing Tmod1 and Tmod4. The γcyto-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γcyto-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3–γcyto-actin–sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca2+ release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γcyto-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4–6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12–15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγcharge and L-type Ca2+current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a ∼17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K D = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

Genome wide association studies (GWAS) identified the ANK1 gene as a common type 2 diabetes mellitus (T2D) susceptibility locus. More recently, GWAS studies identified a novel SNP associated to T2D susceptibility, namely rs508419, in the internal promoter of ANK1 gene, which drives the expression of small ankyrin 1.5 (sAnk1.5), a striated muscle-specific protein localized in the sarcoplasmic reticulum (SR) membrane. sAnk1.5 interacts with the giant sarcomeric protein obscurin, allowing the correct localization of SR around the myofibrils. The rs508419 SNP is characterized by the substitution of a thymine with a cytosine, which determines an increased transcriptional activity of the ANK1 internal promoter, resulting in high protein levels of sAnk1.5 in skeletal muscle biopsies of individuals carrying the C/C variant of the SNP, with respect to individuals carrying either T/T or C/T genotype. Interestingly, the sequence of microRNA-486 (miR-486), a small non-coding RNA, is positioned in the intron between exon 41 and exon 42 of the coding sequence of sAnk1.5. Therefore, miR-486 is expressed under the transcriptional control of both the principal and of the internal promoters of ANK1. miR-486 plays a role in the regulation of the PI3K/AKT signalling pathway, which regulates several cellular processes, such as growth, cellular proliferation and survival, protein synthesis and degradation, lipid and glucose metabolism. In our laboratory we analysed human skeletal muscle biopsies to evaluate whether miR-486 expression levels were increased in individuals carrying the SNP rs508419. These results showed a significant 3-fold increase of miR-486 expression levels in the skeletal muscle of individuals carrying the C/C variant of the SNP compared to those carrying the T/T variant of the SNP. Given that skeletal muscle is one of the main tissues involved in regulating glucose disposal, the aim of this thesis was to verify whether sAnk1.5 and miR-486 overexpression in mouse skeletal muscle might associate with T2D susceptibility. To this goal, we generated a double transgenic mouse model, (TgsAnk1.5/+//TgmiR486/+), hereafter referred to as double Tg (D-Tg), where the sAnk1.5 coding sequence and the miR-486 sequence are under the transcriptional control of the skeletal muscle-specific rat myosin light chain (MLC) promoter, and of the mouse muscle-specific creatine kinase (CKM) promoter, respectively. Accordingly, the D-Tg mouse was expected to present a skeletal muscle-specific increase of sAnk1.5 and miR-486 expression. Indeed, RT-PCR experiments indicated that the expression of the transgene was restricted to striated muscles and sAnk1.5 and miR-486 mRNAs levels were both robustly increased in transgenic mice compared to WT. Accordingly, western blot analysis of protein extracts from gastrocnemius, 4 extensor digitorum longus (EDL) and soleus muscles revealed an increase in sAnk1.5 protein levels of 45%, 60% and 35%, respectively, with respect to control mice. Notably, in the D-Tg mouse model we observed a significant discrepancy between the levels of sAnk1.5 protein expression and the levels of the corresponding mRNA. Indeed, in the gastrocnemius muscle, sAnk1.5 mRNA was about 12 times more expressed in transgenic mice, compared to only 1.5-fold increase in protein expression, suggesting a post-translational regulation of sAnk1.5 expression. Evidence of this was confirmed by muscular administration of the proteasome inhibitor MG132 in transgenic mice. Results showed sAnk1.5 protein expression levels significantly increased of about 35% compared to those observed in the contralateral untreated gastrocnemius muscles. To evaluate the potential association between sAnk1.5 and miR-486 overexpression with T2D, glucose and insulin tolerance were monitored during a period of 12 months in male transgenic mice. Blood glucose levels after overnight fasting were significantly higher in 2 and 6 months old transgenic mice compared to WT controls. However, overall glucose and insulin tolerance was not altered between the two experimental groups. Finally, considering the miR-486 role in regulating PI3K/AKT signalling pathway, which in skeletal muscle fiber is activated following insulin stimulation thus being a key pathway in maintaining glucose homeostasis, we characterized the expression pattern of PTEN, p85α, FOXO1a, pAKTser473, and GLUT4 in the skeletal muscles of D-Tg mice. Currently, we are performing additional experiments where D-Tg mice are fed with a high-fat diet to better evaluate whether sAnk1.5 and miR-486 overexpression might predispose to T2D susceptibility.

Skeletal muscle represents about 40% of the body mass and is the site where the major part of blood glucose is disposed following insulin stimulation. Due to this critical role, skeletal muscle dysfunctions often result in the development of systemic metabolic diseases. Type 2 Diabetes (T2D) is the most common chronic metabolic disorder, representing nearly 90% of the overall diabetes cases. T2D is characterized by insulin resistance followed by reduced insulin release from pancreatic b-cells, resulting in high glucose concentration in bloodstream and glucose intolerance. T2D is a multifactorial disorder, as its onset has both genetic and environmental origins. Genome Wide Association Studies have identified hundreds of single nucleotide polymorphisms (SNPs) associated to T2D susceptibility, in the human genome. Interestingly, several of these SNPs were identified in the ANK1 locus, although these SNPs were found in regions neither coding nor endowed with a regulatory activity. However, two recent independent studies identified a novel SNP in the internal promoter of the ANK1 gene, which drives the expression of sAnk1.5, a striated muscle-specific small ANK1 isoform. The sAnk1.5 protein is localized on the sarcoplasmic reticulum (SR) membrane, in skeletal muscle fibers, and interacts with Obscurin, a giant protein of the sarcomere. This interaction stabilizes the SR and guarantees the close apposition of this organelle around the contractile apparatus. The ANK1 internal promoter carrying the C/C variant displays higher transcriptional activity with respect to the T/T variant. Accordingly, skeletal muscle biopsies of individuals carrying the C/C genotype showed higher levels of both sAnk1.5 mRNA and protein compared to those carrying the T/T genotype. The aim of this thesis was to investigate whether sAnk1.5 overexpression in skeletal muscle might predispose to T2D susceptibility. Accordingly, we generated a transgenic mouse model with the coding sequence of the murine sAnk1.5 under the transcriptional control of the skeletal muscle-specific rat myosin light chain promoter. In these transgenic mice, protein levels of sAnk1.5 were increased up to 50% in skeletal muscles with respect to wild type mice. Basal glucose levels, glucose and insulin tolerance were monitored over a period of 12-months. In addition, 2-months old mice were fed with a high fat diet for twelve weeks. The results obtained did not reveal significant differences in glucose and insulin disposal between transgenic and wild type mice. In conclusion, our results, show that sAnk1.5 overexpression does not appear to predispose to a pre-diabetic or diabetic condition.