Abstract only Myogenesis is a crucial process governing muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the ...increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNML1/DRP1-mediated fragmentation and subsequent removal of mitochondria via p62/SQSTM-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α-mediated biogenesis. Mitochondrial fusion protein OPA1 is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that blocking autophagy with various inhibitors during differentiation results in a blockade in myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.
RATIONALE:Mitochondria play a dual role in the heart, responsible for meeting energetic demands and regulating cell death. Paradigms have held that mitochondrial fission and fragmentation are the ...result of pathologic stresses such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in decreased mitochondrial function and cardiac impairment, suggesting that fission is important for maintaining cardiac and mitochondrial bioenergetic homeostasis.
OBJECTIVE:The purpose of this study is to determine whether mitochondrial fission and fragmentation can be an adaptive mechanism used by the heart to augment mitochondrial and cardiac function during a normal physiologic stress such as exercise.
METHODS AND RESULTS:We demonstrate a novel role for cardiac mitochondrial fission as a normal adaptation to increased energetic demand. During submaximal exercise, 'physiologic' mitochondrial fragmentation results in enhanced, rather than impaired mitochondrial function, and is mediated in-part by β1-adrenergic receptor signaling. Similar to pathologic fragmentation, physiologic fragmentation is induced by activation of Drp1; however, unlike pathologic fragmentation, membrane potential is maintained and regulators of mitophagy are downregulated. Inhibition of fission with P110, Mdivi-1 or in mice with cardiac specific Drp1 ablation, significantly
CONCLUSIONS:These findings demonstrate the requirement for physiological mitochondrial fragmentation to meet the energetic demands of exercise as well as providing additional support for the evolving conceptual framework, where mitochondrial fission and fragmentation play a role in the balance between mitochondrial maintenance of normal physiology and response to disease.
Abstract only Signal transduction through β1 and β2-adrenergic receptors (ARs) is considered a primary mechanism for regulating cardiovascular function and remodeling. Upon β-AR activation (i.e., ...physical activity, cardiac pathology) inotropy and chonotropy increase and mitochondria must quickly meet increased energy demand. This suggests that βARs and mitochondria are coupled mechanistically to rapidly respond to the functional and energetic needs of the heart. To investigate the role of β1 vs. β2-AR signaling on mitochondrial dynamics, we compared β1-/- and β2-/- to WT controls. β2-/- had increased mitochondrial fragmentation (increased number and decreased size) by electron microscopy vs. both WT and β1-/-. β2-/- showed altered regulation of mitochondrial fission: increased Drp1 translocation to the mitochondria vs. WT, whereas β1-/- had lower Drp1 translocation. These data suggest differential regulation of fission by βAR signaling, β1 activating and β2 suppressing fission. Since Ca2+-dependent calcineurin is known to activate Drp1 and Ca2+i is differentially regulated by β-AR signaling, we examined calcineurin as the bridge between β-AR signaling and Drp1 activation. In β2-/-, both Ca2+ transients and calcineurin activity were increased, suggesting β1-AR/Ca2+/calcinurin-mediated fission. To quantify mitochondrial fragmentation and biogenesis, mitotimer-transfected C2C12 cells were treated with the non-specific β-AR agonist isoproterenol resulting in mitochondrial fragmentation that was inhibited by the β1-antagonist CGP 12177 but not by the ß2-antagonist ICI 118551. Taken together, our data indicate that β1 and β2-AR signaling differentially regulate mitochondrial dynamics in the heart through alterations in Ca2+i, leading to calcineurin-induced translocation of Drp1.
Abstract only Mitochondria play a dual role in the heart, responsible for meeting energetic demands and regulating cell death. Current paradigms hold that mitochondrial fission and fragmentation are ...the result of pathologic stresses such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in cardiac impairment, suggesting that fission is important for maintaining normal mitochondrial function. In this study, we identify a novel role for mitochondrial fragmentation as a normal physiological adaptation to increased energetic demand. Using two models of exercise, we demonstrate that “physiologic” mitochondrial fragmentation occurs, results in enhanced mitochondrial function, and is mediated through beta 1-adrenergic receptor signaling. Similar to pathologic fragmentation, physiologic fragmentation is induced by activation of Drp1; however, unlike pathologic fragmentation, membrane potential is maintained and regulators of mitophagy are downregulated. To confirm the role of fragmentation as a physiological adaptation to exercise, we inhibited the pro-fission mediator Drp1 in mice using the peptide inhibitor P110 and had mice undergo exercise. Mice treated with P110 had significantly decreased exercise capacity, decreased fragmentation and inactive Drp1 vs controls. To further confirm these findings, we generated cardiac-specific Drp1 KO mice and had them undergo exercise. Mice with cardiac specific Drp1 KO had significantly decreased exercise capacity and abnormally large mitochondria compared to controls. These findings indicate the requirement for physiological mitochondrial fragmentation to meet the energetic demands of exercise and support the still evolving conceptual framework, where fragmentation plays a role in the balance between mitochondrial maintenance of normal physiology and response to disease.
Abstract
Coral reef health is in rapid decline worldwide yet the molecular mechanisms behind coral death remain poorly understood. The Tumor Necrosis Factor (TNF) receptor-ligand superfamily ...(TNFRSF/TNFSF) is a central mediator of apoptosis and it is hypothesized that the expansion of the TNFRSF/TNFSF occurred following the divergence of invertebrates and vertebrates. Here we challenge this hypothesis and identify more putative coral TNFRSF members than any organism described thus far, including humans. We then predicted Human TNFα (HuTNFα), a known inducer of apoptosis in humans, would also cause apoptosis in coral. Upon HuTNFα stimulation the coral proteome underwent an acidic shift, suggesting the induction signaling cascades. Stimulation of coral with HuTNFα also induced apoptotic blebbing, caspase activation and coral bleaching. This work identifies the first ligand/receptor system to be directly involved with apoptosis and bleaching in coral, and provides evidence for an ancient origin of the TNFRSF/TNFSF that has been functionally maintained for over 550 million years.
The SDF-1α/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate ...SDF-1α and CXCR4 expression in fetal pancreas. We have found that SDF-1α and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) γ-nonobese diabetic mouse. We show that SDF-1α stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1α on ductal cell migration. Importantly, blocking the SDF-1α/CXCR4 axis in IFNγ-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1α-CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.
Full text
Available for:
BFBNIB, NUK, PNG, UL, UM, UPUK
The COP9 Signalosome (CSN) is a highly conserved eight subunit protein complex associated with a wide range of essential biological functions in eukaryotic cells, and directly involved in processes ...including deneddylation, phosphorylation, and ubiquitination. Despite its significant role, very few studies have been undertaken to reveal the interactions between the CSN and its binding partners, and none in human T cells. Here we present a purification method for the CSN and binding proteins via the Streptavidin-Binding Peptide (SBP) fused to CSN Subunit 1 (CSN1). Using this method, coupled with liquid chromatography-mass spectrometry analysis, we identified all eight subunits of the CSN, as well as expected and putative novel binding partners such as a tumor suppressor under the control of Cullin4a-ligase complex; Neurofibromin 2 (Merlin). This work presents a method for fast, reliable, and specific affinity-based purification of a protein complex from a nonadherent cell line. The purification of the CSN and binding partners from T cells can elucidate the roles of CSN in a cell type where it has never been studied before. This proteomic-based approach can broaden our understanding of the functions of the CSN in contexts such as viral-host interactions or immune activation in their natural milieu.
Abstract
Mucosal surfaces serve as a primary entry point for multiple pathogens and are therefore principal sites of immune defense. Here we demonstrate through in vitro and in silico studies that ...increased phage adherence to the host mucosal layer, provides a novel immune defense mechanism. We show that compared to the surrounding environment, phage-to-bacteria ratios were increased on all mucosal surfaces sampled ranging from cnidarians to humans. This increased phage abundance protects the underlying epithelium from bacterial infection. Enrichment of phage on mucus occurs via interactions between host mucin glycoproteins and phage immunoglobulin-like protein domains exposed on phage capsids. Metagenomic analysis found these immunoglobulin-like proteins present in many environments, particularly those adjacent to mucosal surfaces. Preliminary glycan microarrays and 2D gel electrophoresis show that phage adherence can rapidly adapt to hosts mucus glycan profiles, and in response, the host may regulate its mucus glycosylation to select for a beneficial phage community. This adaptation between phage and host provide a mechanism for the manipulation and selection of the mucosal microbiota. Based on these observations, we present the Bacteriophage Adherence to Mucus (BAM) model describing a phage-derived mucosal immunity with potential applicability to all mucosal surfaces, thus opening a novel field of immunological study.
BackgroundPARIS (Parkin Interacting Substrate) is a recently identified zinc finger protein acting as a transcriptional inhibitor for PGC-1α. Previous studies showed that Parkin ubiquitinates PARIS ...and thus marks it for degradation. However, the role of PARIS in the heart has not been studied. The objective of this study is to elucidate the role of PARIS in the context of mitochondrial biogenesis after myocardial ischemia or ischemia/reperfusion.MethodsParkin knockout (PKO) mice and wild-type (WT) C57BL/6J littermates were subjected to 15 mins ischemia followed by 30 mins reperfusion (I/R). Ischemic and non-ischemic region were carefully dissected under microscope and protein fractionation for cytosol, mitochondria and nucleus was performed. For in vitro simulated ischemia (sI) /reperfusion, HL-1 cardiomyocyte in which PARIS was overexpressed or silenced were subjected to 1 hr ischemia or followed by reperfusion. PGC1α and COX4, markers of mitochondrial biogenesis, were measured by RT-PCR and western blot.ResultsOur results show that HL-1 cells subjected to sI showed a decrease in PARIS and an increase in markers of mitochondrial biogenesis. Overexpression of PARIS suppressed the upregulation of PGC1α after sI. Knockdown of PARIS via siRNA did not alter markers of mitochondrial biogenesis under basal conditions, but increased mitochondrial biogenesis 24h after recovery from ischemia. In WT mouse hearts, PGC1α mRNA increased after I/R but not in PKO hearts. Similarly, COX4 protein increased in WT mice after I/R but not in PKO mice. PARIS abundance decreased after I/R in WT mouse heart but not PKO. Furthermore, we found that PARIS is modified by SUMO (Small Ubiquitin-related Modifier) under basal conditions in WT mice. Finally, we noted that SENP5, a de-SUMOylase, increased with I/R in WT mice.ConclusionsThese results suggest that SUMOylation of PARIS may mediate nuclear translocation resulting in suppression of PGC1α transcription under basal conditions, followed by de-repression of PGC1α during I/R. Parkin plays a role in regulation of PARIS and mitochondrial biogenesis in response to ischemic stress.
PDF
