Exploring Obscurin and SPEG Kinase Biology.
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ABSTRACT:
SUBMITTER: Fleming JR
PROVIDER: S-EPMC7957886 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Obscurins comprise a family of giant (~870- to 600-kDa) and small (~250- to 55-kDa) proteins that play important roles in myofibrillogenesis, cytoskeletal organization, and cell adhesion and are implicated in hypertrophic cardiomyopathy and tumorigenesis. Giant obscurins are composed of tandem structural and signaling motifs, including 2 serine/threonine kinase domains, SK1 and SK2, present at the COOH terminus of giant obscurin-B. Using biochemical and cellular approaches, we show for the first time that both SK1 and SK2 possess enzymatic activities and undergo autophosphorylation. SK2 can phosphorylate the cytoplasmic domain of N-cadherin, a major component of adherens junctions, and SK1 can interact with the extracellular domain of the ?1-subunit of the Na(+)/K(+)-ATPase, which also resides in adherens junctions. Immunostaining of nonpermeabilized myofibers and cardiocytes revealed that some obscurin kinase isoforms localize extracellularly. Quantification of the exofacial expression of obscurin kinase proteins indicated that they occupy ~16 and ~5% of the sarcolemmal surface in myofibers and cardiocytes, respectively. Treatment of heart lysates with peptide-N-glycosidase F revealed that while giant obscurin-B localizes intracellularly, possessing dual kinase activity, a small obscurin kinase isoform that contains SK1 localizes extracellularly, where it undergoes N-glycosylation. Collectively, our studies demonstrate that the obscurin kinase domains are enzymatically active and may be involved in the regulation of cell adhesion.
| S-EPMC3633816 | biostudies-literature
Project description:Mutations in striated preferentially expressed protein kinase (SPEG), a member of the myosin light chain kinase protein family, are associated with centronuclear myopathy (CNM), cardiomyopathy, or a combination of both. Burgeoning evidence suggests that SPEG plays critical roles in the development, maintenance, and function of skeletal and cardiac muscles. Here we review the genotype-phenotype relationships and the molecular mechanisms of SPEG-related diseases. This review will focus on the progress made toward characterizing SPEG and its interacting partners, and its multifaceted functions in muscle regeneration, triad development and maintenance, and excitation-contraction coupling. We will also discuss future directions that are yet to be investigated including understanding of its tissue-specific roles, finding additional interacting proteins and their relationships. Understanding the basic mechanisms by which SPEG regulates muscle development and function will provide critical insights into these essential processes and help identify therapeutic targets in SPEG-related disorders.
| S-EPMC8199188 | biostudies-literature
Project description:Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min-1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.
| S-EPMC10967385 | biostudies-literature
Project description:Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections.
| S-EPMC1885211 | biostudies-literature
Project description:The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for five years. Furthermore, genome-wide single nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Cellular-level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.
2024-08-12 | GSE274641 | GEO
Project description:RationaleJunctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear.ObjectiveTo determine the role of SPEG in healthy and failing adult hearts.Methods and resultsProteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice.ConclusionsThe novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.
| S-EPMC5218854 | biostudies-literature
Project description:The aim of the study was mycological examination of ulcerated corneal tissues from an ophthalmic patient. Tissue fragments were analyzed on potato-glucose agar (PDA) and maltose (MA) (Difco) media using standard laboratory techniques. Cultures were identified using classical and molecular methods. Macro- and microscopic colony morphology was characteristic of fungi from the genus Aspergillus (restricted growth series), most probably Aspergillus penicillioides Speg. Molecular analysis of the following rDNA regions: ITS1, ITS2, 5.8S, 28S rDNA, LSU and β-tubulin were carried out for the isolates studied. A high level of similarity was found between sequences from certain rDNA regions, i.e. ITS1-5.8S-ITS2 and LSU, what confirmed the classification of the isolates to the species A. penicillioides. The classification of our isolates to A. penicillioides species was confirmed also by the phylogenetic analysis.The aim of the study was mycological examination of ulcerated corneal tissues from an ophthalmic patient. Tissue fragments were analyzed on potato-glucose agar (PDA) and maltose (MA) (Difco) media using standard laboratory techniques. Cultures were identified using classical and molecular methods. Macro- and microscopic colony morphology was characteristic of fungi from the genus Aspergillus (restricted growth series), most probably Aspergillus penicillioides Speg. Molecular analysis of the following rDNA regions: ITS1, ITS2, 5.8S, 28S rDNA, LSU and β-tubulin were carried out for the isolates studied. A high level of similarity was found between sequences from certain rDNA regions, i.e. ITS1-5.8S-ITS2 and LSU, what confirmed the classification of the isolates to the species A. penicillioides. The classification of our isolates to A. penicillioides species was confirmed also by the phylogenetic analysis.
| S-EPMC7256836 | biostudies-literature
Project description:The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and left ventricular non-compaction (LVNC), have been frequently associated with mutations in sarcomeric proteins. In recent years, advances in DNA sequencing technology has allowed the study of the giant proteins of the sarcomere, such as titin and nebulin. Obscurin has been somewhat neglected in these studies, largely because its functional role is far from clear, although there was an isolated report in 2007 of obscurin mutations associated with HCM. Recently, whole exome sequencing methodology (WES) has been used to address mutations in OBSCN, the gene for obscurin, and OBSCN variants were found to be relatively common in inherited cardiomyopathies. In different studies, 5 OBSCN unique variants have been found in a group of 30 end-stage failing hearts, 6 OBSCN unique variants in 74 HCM cases and 3 OBSCN unique variants in 10 LVNC patients. As yet, the number of known potentially disease-causing OBSCN variants is quite small. The reason for this is that mutations in the OBSCN gene have not been recognised as potentially disease-causing until recently, and were not included in large-scale genetic surveys. OBSCN mutations may be causative of HCM, DCM and LVNC and other cardiomyopathies, or they may work in concert with other variants in the same or other genes to initiate the pathology. Currently, the function of obscurin is not well understood, but we anticipate that many more OBSCN variants linked to cardiomyopathy will be found when the large cohorts of patient sequences available are tested. It is to be hoped that the establishment of the importance of obscurin in pathology will stimulate a thorough investigation of obscurin function.
| S-EPMC5498328 | biostudies-other
Project description:Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.
| S-EPMC7734599 | biostudies-literature
Project description:Nature has developed an exquisite array of methods to introduce halogen atoms into organic compounds. Most of these enzymes are oxidative and require either hydrogen peroxide or molecular oxygen as a cosubstrate to generate a reactive halogen atom for catalysis. Vanadium-dependent haloperoxidases contain a vanadate prosthetic group and utilize hydrogen peroxide to oxidize a halide ion into a reactive electrophilic intermediate. These metalloenzymes have a large distribution in nature, where they are present in macroalgae, fungi, and bacteria, but have been exclusively characterized in eukaryotes. In this minireview, we highlight the chemistry and biology of vanadium-dependent haloperoxidases from fungi and marine algae and the emergence of new bacterial members that extend the biological function of these poorly understood halogenating enzymes.
| S-EPMC2707250 | biostudies-literature