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Pseudokinases are catalytically inactive proteins in the human genome that lack the ability to transfer phosphate from ATP to their substrates. The Tribbles family of pseudokinases contains three members: Tribbles 1, 2, and 3. Tribbles 1 has recently gained importance because of its involvement in various diseases, including cancer. It acts as a scaffolding protein that brings about the degradation of its substrate proteins, such as C/EBPα/ß, MLXIPL, and RAR/RXRα, among others, via the ubiquitin proteasome system. It also serves as an adapter protein, which sequesters different protein molecules and activates their downstream signaling, leading to processes, such as cell survival, cell proliferation, and lipid metabolism. It has been implicated in cancers such as AML, prostate cancer, breast cancer, CRC, HCC, and glioma, where it activates oncogenic signaling pathways such as PI3K-AKT and MAPK and inhibits the anti-tumor function of p53. TRIB1 also causes treatment resistance in cancers such as NSCLC, breast cancer, glioma, and promyelocytic leukemia. All these effects make TRIB1 a potential drug target. However, the lack of a catalytic domain renders TRIB1 "undruggable", but knowledge about its structure, conformational changes during substrate binding, and substrate binding sites provides an opportunity to design small-molecule inhibitors against specific TRIB1 interactions.
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GBM (Glioblastoma) is the most lethal CNS (Central nervous system) tumor in adults, which inevitably develops resistance to standard treatments leading to recurrence and mortality. TRIB1 is a serine/threonine pseudokinase which functions as a scaffold platform that initiates degradation of its substrates like C/EBPα through the ubiquitin proteasome system and also activates MEK and Akt signaling. We found that increased TRIB1 gene expression associated with worse overall survival of GBM patients across multiple cohorts. Importantly, overexpression of TRIB1 decreased RT/TMZ (radiation therapy/temozolomide)-induced apoptosis in patient derived GBM cell lines in vitro. TRIB1 directly bound to MEK and Akt and increased ERK and Akt phosphorylation/activation. We also found that TRIB1 protein expression was maximal during G2/M transition of cell cycle in GBM cells. Furthermore, TRIB1 bound directly to HDAC1 and p53. Importantly, mice bearing TRIB1 overexpressing tumors had worse overall survival. Collectively, these data suggest that TRIB1 induces resistance of GBM cells to RT/TMZ treatments by activating the cell proliferation and survival pathways thus providing an opportunity for developing new targeted therapeutics.
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Neoplasias Encefálicas , Glioblastoma , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Temozolomida/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Apoptose/genética , Quinases de Proteína Quinase Ativadas por Mitógeno , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologiaRESUMO
Purpose: Increasing evidence suggests that ultra-high-dose-rate (UHDR) radiation could result in similar tumor control as conventional (CONV) radiation therapy (RT) while reducing toxicity to surrounding healthy tissues. Considering that radiation toxicity to gonadal tissues can cause hormone disturbances and infertility in young patients with cancer, the purpose of this study was to assess the possible role of UHDR-RT in reducing toxicity to healthy gonads in mice compared with CONV-RT. Methods and Materials: Radiation was delivered to the abdomen or pelvis of female (8 or 16 Gy) and male (5 Gy) C57BL/6J mice, respectively, at conventional (â¼0.4 Gy/s) or ultrahigh (>100 Gy/s) dose rates using an IntraOp Mobetron linear accelerator. Organ weights along with histopathology and immunostaining of irradiated gonads were used to compare toxicity between radiation modalities. Results: CONV-RT and UHDR-RT induced a similar decrease in uterine weights at both studied doses (â¼50% of controls), which indicated similarly reduced ovarian follicular activity. Histologically, ovaries of CONV- and UHDR-irradiated mice exhibited a comparable lack of follicles. Weights of CONV- and UHDR-irradiated testes were reduced to â¼30% of controls, and the percentage of degenerate seminiferous tubules was also similar between radiation modalities (â¼80% above controls). Pairwise comparisons of all quantitative data indicated statistical significance between irradiated (CONV or UHDR) and control groups (from P ≤ .01 to P ≤ .0001) but not between radiation modalities. Conclusions: The data presented here suggest that the short-term effects of UHDR-RT on the mouse gonads are comparable to those of CONV-RT.
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Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder characterized by fibro-fatty infiltration with an increased propensity for ventricular arrhythmias and sudden death. Genetic variants in desmosomal genes are associated with ACM. Incomplete penetrance is a common feature in ACM families, complicating the understanding of how external stressors contribute towards disease development. To analyze the dual role of genetics and external stressors on ACM progression, we developed one of the first mouse models of ACM that recapitulates a human variant by introducing the murine equivalent of the human R451G variant into endogenous desmoplakin (DspR451G/+). Mice homozygous for this variant displayed embryonic lethality. While DspR451G/+ mice were viable with reduced expression of DSP, no presentable arrhythmogenic or structural phenotypes were identified at baseline. However, increased afterload resulted in reduced cardiac performance, increased chamber dilation, and accelerated progression to heart failure. In addition, following catecholaminergic challenge, DspR451G/+ mice displayed frequent and prolonged arrhythmic events. Finally, aberrant localization of connexin-43 was noted in the DspR451G/+ mice at baseline, becoming more apparent following cardiac stress via pressure overload. In summary, cardiovascular stress is a key trigger for unmasking both electrical and structural phenotypes in one of the first humanized ACM mouse models.
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Displasia Arritmogênica Ventricular Direita , Animais , Arritmias Cardíacas/genética , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/metabolismo , Desmoplaquinas/genética , Modelos Animais de Doenças , Coração , Humanos , Camundongos , FenótipoRESUMO
Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/-). ZO-1+/- mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/- mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/- mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/- cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.
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Miocárdio , Junções Íntimas , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Sódio/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation 'hot-spot' within the NH2-terminal third of the DSP protein (specifically, residues 299-515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447-451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation-tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This 'molecular band-aid' provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders.
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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.
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Diferenciação Celular , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/biossíntese , Miócitos Cardíacos/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , Miócitos Cardíacos/citologiaRESUMO
Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder with variable genetic etiologies. Here we focused on understanding the precise molecular pathology of a single clinical variant in DSP, the gene encoding desmoplakin. We initially identified a novel missense desmoplakin variant (p.R451G) in a patient diagnosed with biventricular ACM. An extensive single-family ACM cohort was assembled, revealing a pattern of coinheritance for R451G desmoplakin and the ACM phenotype. An in vitro model system using patient-derived induced pluripotent stem cell lines showed depressed levels of desmoplakin in the absence of abnormal electrical propagation. Molecular dynamics simulations of desmoplakin R451G revealed no overt structural changes, but a significant loss of intramolecular interactions surrounding a putative calpain target site was observed. Protein degradation assays of recombinant desmoplakin R451G confirmed increased calpain vulnerability. In silico screening identified a subset of 3 additional ACM-linked desmoplakin missense mutations with apparent enhanced calpain susceptibility, predictions that were confirmed experimentally. Like R451G, these mutations are found in families with biventricular ACM. We conclude that augmented calpain-mediated degradation of desmoplakin represents a shared pathological mechanism for select ACM-linked missense variants. This approach for identifying variants with shared molecular pathologies may represent a powerful new strategy for understanding and treating inherited cardiomyopathies.
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Arritmias Cardíacas/genética , Calpaína/metabolismo , Cardiomiopatias/genética , Desmoplaquinas/metabolismo , Predisposição Genética para Doença/genética , Mutação , Adulto , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Calpaína/farmacologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Desmoplaquinas/antagonistas & inibidores , Desmoplaquinas/química , Feminino , Glicina , Coração , Insuficiência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Proteínas Recombinantes , Células-TroncoRESUMO
Proper cardiac function requires the synchronous mechanical and electrical coupling of individual cardiomyocytes. The intercalated disc (ID) mediates coupling of neighboring myocytes through intercellular signaling. Intercellular communication is highly regulated via intracellular signaling, and signaling pathways originating from the ID control cardiomyocyte remodeling and function. Herein, we present an overview of the inter- and intracellular signaling that occurs at and originates from the intercalated disc in normal physiology and pathophysiology. This review highlights the importance of the intercalated disc as an integrator of signaling events regulating homeostasis and stress responses in the heart and the center of several pathophysiological processes mediating the development of cardiomyopathies.
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The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFß1). While TGFß1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFß1 were generated to model the observed increase in activity in the failing heart. Activation of TGFß1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFß1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFß1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFß1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFß1 activity in the human cardiac ventricle. Overall, we found that TGFß1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFß1 and reduce heart failure severity.
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Antígeno CD56/metabolismo , Miocárdio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Adesão Celular , Eletrocardiografia , Feminino , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Camundongos Transgênicos , Miocárdio/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Ratos , Disfunção VentricularRESUMO
Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca2+-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysftm1Kcam/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca2+-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca2+-dependent pathway known to be important for sarcolemmal membrane repair.
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Proteínas de Transporte/uso terapêutico , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Proteínas Recombinantes/uso terapêutico , Animais , Modelos Animais de Doenças , Disferlina/deficiência , Disferlina/genética , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Proteínas com Motivo TripartidoRESUMO
The obscurin family of polypeptides is essential for normal striated muscle function and contributes to the pathogenesis of fatal diseases, including cardiomyopathies and cancers. The single mammalian obscurin gene, OBSCN, gives rise to giant (â¼800 kDa) and smaller (â¼40-500 kDa) proteins that are composed of tandem adhesion and signaling motifs. Mammalian obscurin proteins are expressed in a variety of cell types, including striated muscles, and localize to distinct subcellular compartments where they contribute to diverse cellular processes. Obscurin homologs in Caenorhabditis elegans and Drosophila possess a similar domain architecture and are also expressed in striated muscles. The long sought after question, "what does obscurin do?" is complex and cannot be addressed without taking into consideration the subcellular distribution of these proteins and local isoform concentration. Herein, we present an overview of the functions of obscurins and begin to define the intricate relationship between their subcellular distributions and functions in striated muscles.
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Research over the last decade strengthened the understanding that skeletal muscles are not only the major tissue in the body from a volume point of view but also function as a master regulator contributing to optimal organismal health. These new contributions to the available body of knowledge triggered great interest in the roles of skeletal muscle beyond contraction. The World Health Organization, through its Global Burden of Disease (GBD) report, recently raised further awareness about the key importance of skeletal muscles as the GDB reported musculoskeletal (MSK) diseases have become the second greatest cause of disability, with more than 1.7 billion people in the globe affected by a diversity of MSK conditions. Besides their role in MSK disorders, skeletal muscles are also seen as principal metabolic organs with essential contributions to metabolic disorders, especially those linked to physical inactivity. In this review, we have focused on the unique function of new genes/proteins (i.e., MTMR14, MG29, sarcalumenin, KLF15) that during the last few years have helped provide novel insights about muscle function in health and disease, muscle fatigue, muscle metabolism, and muscle aging. Next, we provide an in depth discussion of how these genes/proteins converge into a common function of acting as regulators of intracellular calcium homeostasis. A clear link between dysfunctional calcium homeostasis is established and the special role of store-operated calcium entry is analyzed. The new knowledge that has been generated by the understanding of the roles of previously unknown modulatory genes of the skeletal muscle excitation-contraction coupling (ECC) process brings exciting new possibilities for treatment of MSK diseases, muscle regeneration, and skeletal muscle tissue engineering. The next decade of skeletal muscle and MSK research is bound to bring to fruition applied knowledge that will hopefully offset the current heavy and sad burden of MSK diseases on the planet.