Sphingosine-1 Phosphate Lyase Regulates Sensitivity of Pancreatic Beta-Cells to Lipotoxicity

Elevated levels of free fatty acids (FFAs) have been related to pancreatic beta-cell failure in type 2 diabetes (T2DM), though the underlying mechanisms are not yet fully understood. FFAs have been shown to dysregulate formation of bioactive sphingolipids, such as ceramides and sphingosine-1 phosphate (S1P) in beta-cells. The aim of this study was to analyze the role of sphingosine-1 phosphate lyase (SPL), a key enzyme of the sphingolipid pathway that catalyzes an irreversible degradation of S1P, in the sensitivity of beta-cells to lipotoxicity. To validate the role of SPL in lipotoxicity, we modulated SPL expression in rat INS1E cells and in human EndoC-βH1 beta-cells. SPL overexpression in INS1E cells (INS1E-SPL), which are characterized by a moderate basal expression level of SPL, resulted in an acceleration of palmitate-mediated cell viability loss, proliferation inhibition and induction of oxidative stress. SPL overexpression affected the mRNA expression of ER stress markers and mitochondrial chaperones. In contrast to control cells, in INS1E-SPL cells no protective effect of oleate was detected. Moreover, Plin2 expression and lipid droplet formation were strongly reduced in OA-treated INS1E-SPL cells. Silencing of SPL in human EndoC-βH1 beta-cells, which are characterized by a significantly higher SPL expression as compared to rodent beta-cells, resulted in prevention of FFA-mediated caspase-3/7 activation. Our findings indicate that an adequate control of S1P degradation by SPL might be crucially involved in the susceptibility of pancreatic beta-cells to lipotoxicity.

Mitochondrial Chaperones and Proteases in Cardiomyocytes and Heart Failure

Heart failure is one of the leading causes of morbidity and mortality worldwide. In cardiomyocytes, mitochondria are not only essential organelles providing more than 90% of the ATP necessary for contraction, but they also play critical roles in regulating intracellular Ca2+ signaling, lipid metabolism, production of reactive oxygen species (ROS), and apoptosis. Because mitochondrial DNA only encodes 13 proteins, most mitochondrial proteins are nuclear DNA-encoded, synthesized, and transported from the cytoplasm, refolded in the matrix to function alone or as a part of a complex, and degraded if damaged or incorrectly folded. Mitochondria possess a set of endogenous chaperones and proteases to maintain mitochondrial protein homeostasis. Perturbation of mitochondrial protein homeostasis usually precedes disruption of the whole mitochondrial quality control system and is recognized as one of the hallmarks of cardiomyocyte dysfunction and death. In this review, we focus on mitochondrial chaperones and proteases and summarize recent advances in understanding how these proteins are involved in the initiation and progression of heart failure.

Heat Shock Proteins in Glioblastoma Biology: Where Do We Stand?

Heat shock proteins (HSPs) are evolutionary conserved proteins that work as molecular chaperones and perform broad and crucial roles in proteostasis, an important process to preserve the integrity of proteins in different cell types, in health and disease. Their function in cancer is an important aspect to be considered for a better understanding of disease development and progression. Glioblastoma (GBM) is the most frequent and lethal brain cancer, with no effective therapies. In recent years, HSPs have been considered as possible targets for GBM therapy due their importance in different mechanisms that govern GBM malignance. In this review, we address current evidence on the role of several HSPs in the biology of GBMs, and how these molecules have been considered in different treatments in the context of this disease, including their activities in glioblastoma stem-like cells (GSCs), a small subpopulation able to drive GBM growth. Additionally, we highlight recent works that approach other classes of chaperones, such as histone and mitochondrial chaperones, as important molecules for GBM aggressiveness. Herein, we provide new insights into how HSPs and their partners play pivotal roles in GBM biology and may open new therapeutic avenues for GBM based on proteostasis machinery.

The mitochondrial HSP90 paralog TRAP1 forms an OXPHOS-regulated tetramer and is involved in maintaining mitochondrial metabolic homeostasis

BackgroundThe molecular chaperone TRAP1, the mitochondrial isoform of cytosolic HSP90, remains poorly understood with respect to its pivotal role in the regulation of mitochondrial metabolism. Most studies have found it to be an inhibitor of mitochondrial oxidative phosphorylation (OXPHOS) and an inducer of the Warburg phenotype of cancer cells. However, others have reported the opposite and there is no consensus on the relevant TRAP1 interactors. This calls for a more comprehensive analysis of the TRAP1 interactome and of how TRAP1 and mitochondrial metabolism mutually affect each other.ResultsWe show that the disruption of the gene for TRAP1 in a panel of cell lines dysregulates OXPHOS by a metabolic rewiring that induces the anaplerotic utilization of glutamine metabolism to replenish TCA cycle intermediates. Restoration of wild-type levels of OXPHOS requires full-length TRAP1. Whereas the TRAP1 ATPase activity is dispensable for this function, it modulates the interactions of TRAP1 with various mitochondrial proteins. Quantitatively by far the major interactors of TRAP1 are the mitochondrial chaperones mtHSP70 and HSP60. However, we find that the most stable stoichiometric TRAP1 complex is a TRAP1 tetramer, whose levels change in response to both a decline or an increase in OXPHOS.ConclusionsOur work provides a roadmap for further investigations of how TRAP1 and its interactors such as the ATP synthase regulate cellular energy metabolism. Our results highlight that TRAP1 function in metabolism and cancer cannot be understood without a focus on TRAP1 tetramers as potentially the most relevant functional entity.

Structural comparison of yeast and human intra-mitochondrial lipid transport systems

Mitochondria depend on a tightly regulated supply of phospholipids. The protein of relevant evolutionary and lymphoid interest (PRELI)/Ups1 family together with its mitochondrial chaperones [TP53-regulated inhibitor of apoptosis 1 (TRIAP1)/Mdm35] represents a unique heterodimeric lipid-transfer system that is evolutionary conserved from yeast to man. Recent X-ray crystal structures of the human and yeast systems are compared and discuss here and shed new insight into the mechanism of the PRELI/Ups1 system.

Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity

Heat-shock response is an adaptive response to proteotoxic stresses including heat shock, and is regulated by heat-shock factor 1 (HSF1) in mammals. Proteotoxic stresses challenge all subcellular compartments including the mitochondria. Therefore, there must be close connections between mitochondrial signals and the activity of HSF1. Here, we show that heat shock triggers nuclear translocation of mitochondrial SSBP1, which is involved in replication of mitochondrial DNA, in a manner dependent on the mitochondrial permeability transition pore ANT–VDAC1 complex and direct interaction with HSF1. HSF1 recruits SSBP1 to the promoters of genes encoding cytoplasmic/nuclear and mitochondrial chaperones. HSF1–SSBP1 complex then enhances their induction by facilitating the recruitment of a chromatin-remodelling factor BRG1, and supports cell survival and the maintenance of mitochondrial membrane potential against proteotoxic stresses. These results suggest that the nuclear translocation of mitochondrial SSBP1 is required for the regulation of cytoplasmic/nuclear and mitochondrial proteostasis against proteotoxic stresses.

Mitochondrial Heat Shock Protein Machinery Hsp70/Hsp40 Is Indispensable for Proper Mitochondrial DNA Maintenance and Replication

ABSTRACT Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import. IMPORTANCE Impairment or loss of mitochondrial DNA is associated with mitochondrial dysfunction and a wide range of neural, muscular, and other diseases. We present the first evidence showing that the entire mtHsp70/mtHsp40 machinery plays an important role in mitochondrial DNA replication and maintenance, a function likely retained from prokaryotes. These abundant, ubiquitous, and multifunctional chaperones share phenotypes with enzymes engaged in the initial stages of replication of the mitochondrial DNA in T. brucei.