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Therapy resistance to a selective B-Raf inhibitor (BRAFi) poses a challenge in treating patients with BRAF-mutant melanomas. Here, we report that RNA interference of mortalin (HSPA9/GRP75), a mitochondrial molecular chaperone often upregulated and mislocalized in melanoma, can effectively induce death of vemurafenib-resistant progenies of human B-RafV600E melanoma cell lines, A375 and Colo-829. Mortalin depletion induced death of vemurafenib-resistant cells at similar efficacy as observed in vemurafenib-naive parental cells. This lethality was correlated with perturbed mitochondrial permeability and was attenuated by knockdown of adenine nucleotide translocase (ANT) and cyclophilin D (CypD), the key regulators of mitochondrial permeability. Chemical inhibition of MEK1/2 and ERK1/2 also suppressed mortalin depletion-induced death and mitochondrial permeability in these cells. These data suggest that mortalin and MEK/ERK regulate an ANT/CypD-associated mitochondrial death mechanism(s) in B-RafV600E melanoma cells and that this regulation is conserved even after these cells develop BRAFi resistance. We also show that doxycycline-induced mortalin depletion can effectively suppress the xenografts of vemurafenib-resistant A375 progeny in athymic nude mice. These findings suggest that mortalin has potential as a candidate therapeutic target for BRAFi-resistant BRAF-mutant tumors.

Mitochondria provide the front-line of defense against the tumor-promoting effects of oxidative stress. Here we show that the prostate-specific homeoprotein, NKX3.1, suppresses prostate cancer initiation by protecting mitochondria from oxidative stress. Integrating analyses of genetically-engineered mouse models, human prostate cancer cells, and human prostate cancer organotypic cultures, we find that, in response to oxidative stress, NKX3.1 is imported to mitochondria via the chaperone protein, HSPA9, where it regulates transcription of mitochondrial-encoded electron transport chain (ETC) genes, thereby restoring oxidative phosphorylation and preventing cancer initiation. Germline polymorphisms of NKX3.1 associated with increased cancer risk fail to protect from oxidative stress or suppress tumorigenicity. Low expression levels of NKX3.1 combined with low expression of mitochondrial ETC genes are associated with adverse clinical outcome, whereas high levels of mitochondrial NKX3.1 protein are associated with favorable outcome. This work reveals an extranuclear role for NKX3.1 in suppression of prostate cancer by protecting mitochondrial function. STATEMENT OF SIGNIFICANCE: Our findings uncover a non-nuclear function for NKX3.1 that is a key mechanism for suppression of prostate cancer. Analyses of the expression levels and sub-cellular localization of NKX3.1 in patients at risk of cancer progression may improve risk assessment in a precision prevention paradigm, particularly for men undergoing active surveillance.

As a cofactor, iron–sulfur (Fe–S) cluster binds to proteins or enzymes that play important roles in various important biological processes, including DNA synthesis and repair, mitochondrial function, gene transcription and translation. In mammals, the core components involved in Fe–S cluster biosynthesis are considered to include the scaffold protein ISCU, cysteine desulfurase NFS1 and its accessory proteins ISD11 and ACP, and frataxin (FXN). Proteins involved in Fe–S cluster transfer have been found to include HSC20/HSPA9, as chaperone system, and Fe–S cluster carriers. The biosynthesis and transfer of Fe–S clusters to Fe–S recipients require fine-tune regulation. Recently, significant progress has been made in the structure and mechanism of mitochondrial Fe–S biosynthesis and transfer. Based on, especially, the development of DNA sequencing technology, bioinformatics, and gene editing technology, diseases caused by mutations of Fe–S cluster-related genes have been revealed in recent years, promoting the rapid development in the field of Fe–S and human health. This review focuses on the function of genes involved in Fe–S cluster biosynthesis and transfer and on the diseases caused by the mutations of the related genes. Finally, some questions we are facing are raised, new hypotheses presented, and the perspectives discussed.

The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.

We combined quantitative proteomics with N terminome profiling and substrate trapping AP-MS to identify new ClpXP substrates. Mitochondrial protein N termini isolated from mouse hearts revealed frequent aminopeptidase processing after MTS cleavage and many internal protease-generated neo-N termini. CLPP-deficient mice showed perturbed processing patterns with overall accumulation of protease-generated neo-N termini. New high-confidence candidate ClpXP substrates were identified by strong changes in N termini abundance or pull-downs with inactive CLPP and validated using immunoblotting and cycloheximide-chase experiments., Graphical Abstract Highlights • Mitochondrial heart N terminome shows aminopeptidase processing after MTS cleavage. • CLPP-deficiency alters protein processing patterns in mouse heart mitochondria. • Candidate substrates identified by N termini accumulation and interaction with inactive ClpXP. • UQCRC1, HSPA9 and OAT validated biochemically as high confidence ClpXP substrates., The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp−/− mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

Increased metabolic flux produces potentially harmful side-products, such as reactive dicarbonyl and oxygen species. The reactive dicarbonly methylglyoxal (MG) can impair oxidative capacity, which is downregulated in type 2 diabetes. Heat shock proteins (HSPs) of subfamily A (Hsp70s) promote ATP-dependent processing of damaged proteins during MG exposure which also involve mitochondrial proteins. Since the protection of mitochondrial proteins could promote higher production of reactive metabolites due to increased substrate flux, tight regulation of HspA-mediated protein handling is important. We hypothesized that stress-inducible HspAs (HspA1A/HspA1B) are pivotal for maintaining mitochondrial biogenesis during acute MG-stress. To analyze the role of stress-inducible HspA1A/HspA1B for maintenance of mitochondrial homeostasis during acute MG exposure, we knocked out HSPA1A/HSPA1B in mouse endothelial cells. HSPA1A/HSPA1B KO cells showed upregulation of the mitochondrial chaperones HspA9 (mitochondrial Hsp70/mortalin) and HspD1 (Hsp60) as well as induction of mitochondrial biogenesis upon MG exposure. Increased mitochondrial biogenesis was reflected by elevated mitochondrial branching, total count and area as well as by upregulation of mitochondrial proteins and corresponding transcription factors. Our findings suggest that mitochondrial HspA9 and HspD1 promote mitochondrial biogenesis during acute MG stress, which is counterregulated by HspA1A/HspA1B to prevent mitochondrial overstimulation and to maintain balanced oxidative capacity under metabolic stress conditions. These data support an important role of HSPs in MG-induced hormesis.

The human HSP70 family is a type of heat shock protein (HSP), consisting of 13 members encoded by the HSPA genes. HSPs play important roles in regulating cellular responses and functions during carcinogenesis, but their relationship with colon cancer is unclear. In our study, we found that the expressions of HSPA1B, HSPA4, HSPA5, HSPA6, HSPA8, HSPA9, HSPA13, and HSPA14 were significantly increased, while those of HSPA1A, HSPA2, HSPA7, and HSPA12B were significantly decreased in colon cancer tissues. The expression of HSPA gene family members was associated with some clinicopathological characteristics, including age, gender, TNM stage, pathological stage, and CEA level. Furthermore, the Kaplan–Meier method and Cox regression analysis showed that high HSPA1A, HSPA1B, and HSPA7 expressions were related to unfavorable survival, and high HSPA9 was associated with favorable survival. The relationships between HSPA1A and HSPA9 expression and survival were validated in the GEO dataset, and the HSPA1A and HSPA9 protein expression differences between colon cancer tissues and normal tissues were validated in the UALCAN database. Methylation of HSPA1A and HSPA9 was also analyzed, and it was found that the methylation of the HSPA1A promoter was significantly increased, and the methylation of the HSPA9 promoter was significantly decreased in colon cancer tissues. Increasing the methylation level of the HSPA1A gene and decreasing the methylation level of HSPA9 were related to favorable prognosis. The expression difference of HSPA1A/HSPA1B/HSPA7/HSPA9 was verified in colon cancer cell lines and colonic epithelial cells. Gene ontology analysis was used to screen signal pathways related to HSPA1A-, HSPA1B-, HSPA7-, and HSPA9- high phenotype. In summary, the increased expressions of HSPA1A1, HSPA1B, and HSPA7 were associated with poor prognosis, while that of HSPA9 was related to favorable prognosis for colon cancer patients.

Background Treatments based on production of reactive oxygen species for bladder cancer such as photodynamic therapy (PDT) have been marginalized due to low specificity and the existence of resistance mainly associated with the up-regulation of Heat Shock Proteins (HSPs). To overcome these barriers, the establishment of strategies combining PDTs with HSP inhibitors may be promising and the identification of HSPs involved with oxidative stress from bladder tumors in animal models represents a key step in this direction. Materials Thus, the present study aims to identify cytosolic and mitochondrial HSPs up expressed in murine bladder tumors and in the urothelial carcinoma cell line MB49 by qRT-PCR screening, and to analyze the importance of the activity of the HSPs associated with oxidative stress protection in the survival of the MB49 using strategy of inhibition in vitro. Results Results showed that both tumor tissues and MB49 cells in culture had significant overexpression of the mitochondrial HSPA9 (mortalin) and HSP60 mRNAs, while the cytosolic HSP90 was overexpressed only in the tumor. The effect of mortalin in the MB49 cells survival under oxidative stress was evaluated in vitro in presence of the specific inhibitor MKT-077 and H2O2. The findings showed that MB49 viability was permanently reduced by the MKT-077 in a dose-dependent manner by inducing apoptosis or necrosis, mainly under oxidative stress conditions. Conclusion Results suggest that mortalin is preferentially expressed in the MB49 cancer model and plays a key role in tumoral survival, especially under oxidative stress, making this HSP a potential target for an alternative treatment combining PDT with HSP inhibitors.

Mitochondria provide the first line of defense against the tumor-promoting effects of oxidative stress. Here we show that the prostate-specific homeoprotein NKX3.1 suppresses prostate cancer initiation by protecting mitochondria from oxidative stress. Integrating analyses of genetically engineered mouse models, human prostate cancer cells, and human prostate cancer organotypic cultures, we find that, in response to oxidative stress, NKX3.1 is imported to mitochondria via the chaperone protein HSPA9, where it regulates transcription of mitochondrial-encoded electron transport chain (ETC) genes, thereby restoring oxidative phosphorylation and preventing cancer initiation. Germline polymorphisms of NKX3.1 associated with increased cancer risk fail to protect from oxidative stress or suppress tumorigenicity. Low expression levels of NKX3.1 combined with low expression of mitochondrial ETC genes are associated with adverse clinical outcome, whereas high levels of mitochondrial NKX3.1 protein are associated with favorable outcome. This work reveals an extranuclear role for NKX3.1 in suppression of prostate cancer by protecting mitochondrial function. Significance: Our findings uncover a nonnuclear function for NKX3.1 that is a key mechanism for suppression of prostate cancer. Analyses of the expression levels and subcellular localization of NKX3.1 in patients at risk of cancer progression may improve risk assessment in a precision prevention paradigm, particularly for men undergoing active surveillance. See related commentary by Finch and Baena, p. 2132. This article is highlighted in the In This Issue feature, p. 2113

The 70 kDa heat shock proteins (Hsp70) are prone to self-assembly under thermal stress conditions, forming supramolecular assemblies (SMA), what may have detrimental consequences for cellular viability. In mitochondria, the cochaperone Hsp70-escort protein 1 (Hep1) maintains mitochondrial Hsp70 (mtHsp70) in a soluble and functional state, contributing to preserving proteostasis. Here we investigated the interaction between human Hep1 (hHep1) and HSPA9 (human mtHsp70) or HSPA1A (Hsp70-1A) in monomeric and thermic SMA states to unveil further information about the involved mechanisms. hHep1 was capable of blocking the formation of HSPA SMAs under a thermic treatment and stimulated HSPA ATPase activity in both monomeric and preformed SMA. The interaction of hHep1 with both monomeric and SMA HSPAs displayed a stoichiometric ratio close to 1, suggesting that hHep1 has access to most protomers within the SMA. Interestingly, hHep1 remodeled HSPA9 and HSPA1A SMAs into smaller forms. Furthermore, hHep1 was detected in the mitochondria and nucleus of cells transfected with the respective coding DNA and interacted with liposomes resembling mitochondrial membranes. Altogether, these new features reinforce that hHep1 act as a "chaperone for a chaperone", which may play a critical role in cellular proteostasis.

We investigated the tyrosinase-associated melanogenesis in melanoma cells by using OMICS techniques. We characterized the chromosome copy numbers, including Chr 11q21 where the tyrosinase gene is located, from several melanoma cell lines (TXM13, G361, and SK-MEL-28) by using array CGH. We revealed that 11q21 is stable in TXM13 cells, which is directly related to a spontaneous high melanin pigment production. Meanwhile, significant loss of copy number of 11q21 was found in G361 and SK-MEL-28. We further profiled the proteome of TXM13 cells by LC-ESI-MSMS and detected more than 900 proteins, then predicted 11 hub proteins (YWHAZ; HSP90AA1; HSPA5; HSPA1L; HSPA9; HSP90B1; HSPA1A; HSPA8; FKSG30; ACTB; DKFZp686DQ972) by using an interactomic algorithm. YWHAZ (25% interaction in the network) is thought to be a most important protein as a linking factor between tyrosinase-triggered melanogenesis and melanoma growth. Bioinformatic tools were further applied for revealing various physiologic mechanisms and functional classification. The results revealed clues for the spontaneous pigmentation capability of TXM13 cells, contrary to G361 and SK-MEL-28 cells, which commonly have depigmentation properties during subculture. Our study comparatively conducted the genome-wide screening and proteomic profiling integrated interactomics prediction for TXM13 cells and suggests new insights for studying both melanogenesis and melanoma.

We investigate in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5/mortalin is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we study an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displays ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of DJ-1b, LRRK, p50, p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the autophagy machinery or overexpression of human mortalin broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects.

Enterovirus A71 (EV-A71) is one of the major etiologic agents causing hand, foot, and mouth disease (HFMD) in children and occasionally causes severe neurological diseases or even death. EV-A71 replicates rapidly in host cells. For a successful infection, viruses produce large quantities of viral proteins in a short period, which requires cellular chaperone proteins for viral protein folding and viral particle assembly. In this study, we explored the roles of the heat shock protein 70 (HSP70) chaperone subnetwork in the EV-A71 life cycle. Our results revealed that EV-A71 exploits multiple HSP70s at each step of the viral life cycle, i.e., viral entry, translation, replication, assembly and release, and that each HSP70 typically functions in several stages of the life cycle. For example, the HSP70 isoforms HSPA1, HSPA8, and HSPA9 are required for viral entry and the translational steps of the infection. HSPA8 and HSPA9 may facilitate folding and stabilize viral proteins 3D and 2C, respectively, thus contributing to the formation of a replication complex. HSPA8 and HSPA9 also promote viral particle assembly, whereas HSPA1 and HSPA8 are involved in viral particle release. Because of the importance of various HSP70s at distinct steps of the viral life cycle, an allosteric inhibitor, JG40, which targets all HSP70s, significantly blocks EV-A71 infection. JG40 also blocks the replication of several other enteroviruses, such as coxsackievirus (CV) A16, CVB1, CVB3, and echovirus 11. Thus, targeting HSP70s may be a means of providing broad-spectrum antiviral therapy.

Mortalin [also known as heat shock protein family A (HSP70) member 9 (HSPA9) or glucose-regulated protein 75 (GRP75)] is a mitochondrial molecular chaperone that is often up-regulated and mislocalized in tumors with abnormal activation of the kinases MEK and ERK. Here, we found that mortalin depletion was selectively lethal to tumor and immortalized normal cells expressing the mutant kinase B-RafV600E or the chimeric protein ΔRaf-1:ER and that MEK-ERK-sensitive regulation of the peptide-binding domain in mortalin was critical to cell survival or death. Proteomics screening identified adenine nucleotide translocase 3 (ANT3) as a previously unknown mortalin substrate and cell survival/death effector. Mechanistically, increased MEK-ERK signaling activity and mortalin function converged opposingly on the regulation of mitochondrial permeability. Specifically, whereas MEK-ERK activity increased mitochondrial permeability by promoting the interaction between ANT3 and the peptidyl-prolyl isomerase cyclophilin D (CypD), mortalin decreased mitochondrial permeability by inhibiting this interaction. Hence, mortalin depletion increased mitochondrial permeability in MEK-ERK-deregulated cells to an extent that triggered cell death. HSP70 inhibitor derivatives that effectively inhibited mortalin suppressed the proliferation of B-RafV600E tumor cells in culture and in vivo, including their B-Raf inhibitor-resistant progenies. These findings suggest that targeting mortalin has potential as a selective therapeutic strategy in B-Raf-mutant or MEK-ERK-driven tumors.

Due to the lack of specific symptoms in early thymic epithelial tumours (TETs), patients are mostly in the advanced stage at the time of presentation. The aim of the present study was to explore the mechanism by which the long noncoding RNA (lncRNA) LOXL1-AS1 affects thymoma and thymic carcinoma progression by targeting the miR-525-5p-HSPA9 axis. Bioinformatics was used to analyse the process of LOXL1-AS1 targeting miR-525-5p-HSPA9 and its expression characteristics in TET. The relationships between LOXL1-AS1, miR-525-5p, HSPA9 and prognosis were analysed. The dual luciferase reporter assay was applied to verify targeting. The gene was knocked down or overexpressed by plasmid transfection. Cell counting kit 8 (CCK-8) assay, flow cytometry and Transwell assay were used to detect cell viability, apoptosis and invasion ability, respectively. Proteins and RNAs were examined by western blot analysis and qPCR, respectively. A tumour-burdened assay was used to perform in vivo verification. LOXL1-AS1 and HSPA9 were overexpressed in thymoma and thymic carcinoma; high levels of LOXL1-AS1 and HSPA9 were associated with poor prognosis, and there was a significant positive correlation between their levels. Downregulation of miR-525-5p expression was also associated with poor prognosis of patients. Clinical trials also demonstrated the same trends. miR-525-5p inhibited the expression of HSPA9 protein by targeting the 3′-untranslated region (UTR) of HSPA9 mRNA. LOXL1-AS1 promoted the expression of HSPA9 as a sponge targeting miR-525-5p. Animal experiment results also showed that knockdown of miR-525-5p promoted cancer by promoting the expression of HSPA9. In conclusion, LOXL1-AS1 and HSPA9 are highly expressed in thymoma and thymic carcinoma; miR-525-5p is expressed at low levels in thymoma and thymic carcinoma; and downregulation of miR-525-5p is associated with poor prognosis. In summary, this study demonstrates that LOXL1-AS1 acts as a sponge that targets miR-525-5p to promote HSPA9 expression, thereby promoting the growth and invasion and inhibiting apoptosis of thymoma and thymic carcinoma cells.

Mitochondria are fundamental structures that fulfil important and diverse functions within cells, including cellular respiration and iron-sulfur cluster biogenesis. Mitochondrial function is reliant on the organelles proteome, which is maintained and adjusted depending on cellular requirements. The majority of mitochondrial proteins are encoded by nuclear genes and must be trafficked to, and imported into the organelle following synthesis in the cytosol. These nuclear-encoded mitochondrial precursors utilise dynamic and multimeric translocation machines to traverse the organelles membranes and be partitioned to the appropriate mitochondrial subcompartment. Yeast model systems have been instrumental in establishing the molecular basis of mitochondrial protein import machines and mechanisms, however unique players and mechanisms are apparent in higher eukaryotes. Here, we review our current knowledge on mitochondrial protein import in human cells and how dysfunction in these pathways can lead to disease.

Heat stress hampers egg production and lowers fertility in layers. This study investigated global protein abundance in the small yellow follicles (SYFs, 6–8 mm diameter) of a broiler-type strain of Taiwan country chickens (TCCs) under acute heat stress. Twelve 30-week-old TCC hens were allocated to a control group maintained at 25°C, and to three acute heat-stressed groups subjected to 38°C for 2 h without recovery, with 2-h recovery, or with 6-h recovery. Two-dimensional difference gel electrophoresis analysis identified 119 significantly differentially expressed proteins after acute heat exposure. Gene ontology analysis revealed that most of these proteins are involved in molecular binding (34%), catalytic activity (23%), and structural molecule activity (11%), and participate in metabolic processes (20%), cellular processes (20%), and cellular component organization or biogenesis (11%). Proteins associated with stress response and survival (HSP25, HSP47, HSP70, HSC70, HSPA9), cytoskeleton remodeling, mitochondrial metabolic process of ATP production, antioxidative defense (peroxiredoxin-6), cargo lipid export and delivery (vitellogenin, apolipoprotein B and A1), and toxin/metabolite clearance and delivery (albumin) were upregulated after acute heat stress in the SYFs of TCCs. No overt cell death and atresia were observed in SYFs after acute heat stress. Collectively, these responses may represent a protective mechanism to maintain follicle cell integrity and survival, thereby ensuring a sufficient pool of SYFs for selection into the ovulation hierarchy for successful egg production.

The genetic basis of stress resistance in extremophilic microalgae is not well studied. In this study, a gene of unknown function, the cluster58 or CL58 gene, was identified from the halotolerant green alga Chlamydomonas W80 and characterized. The CL58 gene encodes a protein containing a domain of unknown function, the CHRD domain, and a putative secretory signaling sequence at its N-terminus. The levels of CL58 mRNA increased in response to high copper levels and low temperatures. When the CL58 gene was heterologously expressed as a fusion gene with the NanoLuc luciferase gene in Chlamydomonas reinhardtii, a majority of the NanoLuc activity was detected in the culture medium compared with that in the intracellular fraction. A mutagenic analysis revealed that the putative secretory signaling sequence was sufficient for the secretion of the CL58-NanoLuc fusion protein. In addition, we expressed the protein encoded by the CL58 gene in Escherichia coli; the recombinant, soluble protein was then purified. In summary, we identified a novel gene from C. W80 that appears to encode a stress-responsive, CHRD domain-containing secreted protein.

Since their discovery, heat shock proteins (HSPs) have been identified in all domains of life, which demonstrates their importance and conserved functional role in maintaining protein homeostasis. Mitochondria possess several members of the major HSP sub-families that perform essential tasks for keeping the organelle in a fully functional and healthy state. In humans, the mitochondrial HSP70 chaperone system comprises a central molecular chaperone, mtHSP70 or mortalin (HSPA9), which is actively involved in stabilizing and importing nuclear gene products and in refolding mitochondrial precursor proteins, and three co-chaperones (HSP70-escort protein 1—HEP1, tumorous imaginal disc protein 1—TID-1, and Gro-P like protein E—GRPE), which regulate and accelerate its protein folding functions. In this review, we summarize the roles of mitochondrial molecular chaperones with particular focus on the human mtHsp70 and its co-chaperones, whose deregulated expression, mutations, and post-translational modifications are often considered to be the main cause of neurological disorders, genetic diseases, and malignant growth.

A variable-length poly-T variant in intron 6 of the TOMM40 gene, rs10524523, is associated with risk and age-of-onset of sporadic (late-onset) Alzheimer's disease. In Caucasians, the three predominant alleles at this locus are Short (S), Long (L) or Very long (VL). On an APOE e3/3 background, the S/VL and VL/VL genotypes are more protective than S/S. The '523 poly-T has regulatory properties, in that the VL poly-T results in higher expression than the S poly-T in luciferase expression systems. The aim of the current work was to identify effects on cellular bioenergetics of increased TOM40 protein expression. MitoTracker Green fluorescence and autophagic vesicle staining was the same in control and over-expressing cells, but TOM40 over-expression was associated with increased expression of TOM20, a preprotein receptor of the TOM complex, the mitochondrial chaperone HSPA9, and PDHE1a, and increased activities of the oxidative phosphorylation complexes I and IV and of the TCA member α-ketoglutaric acid dehydrogenase. Consistent with the complex I findings, respiration was more sensitive to inhibition by rotenone in control cells than in the TOM40 over-expressing cells. In the absence of inhibitors, total cellular ATP, the mitochondrial membrane potential, and respiration were elevated in the over-expressing cells. Spare respiratory capacity was greater in the TOM40 over-expressing cells than in the controls. TOM40 over-expression blocked Ab-elicited decreases in the mitochondrial membrane potential, cellular ATP levels, and cellular viability in the control cells. These data suggest elevated expression of TOM40 may be protective of mitochondrial function.

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

CHRNA7 is a neurodevelopmental protein involved in differentiation and neurogenesis, which is also named as nicotinic acetylcholine receptors, cholinergic receptor, nicotinic, alpha 7 (neuronal). The protein encoded by this gene forms a homo-oligomeric channel. It is a major component of brain nicotinic receptors displays that are blocked by and sensitive to alpha-bungarotoxin. Studies reports involvement of CHRNA7 protein in different neurological diseases. Non-availability of 3-dimensional (3D) structure leads the study toward structure 3D prediction along with its interaction analysis. The current paper is focused on the structure prediction through homology modeling of CHRNA7 along with binding site prediction using Schrödinger software suite. In continuation of the study, protein-protein interaction analysis is carried out by using string database. Tertiary structure along with binding sites was obtained, and visualized CHRAN7 protein have interaction with CHRNA protein family along with JAK2, AKT1, PICK1 protein that are involved in neurological disease. Structure formation analysis is an important aspect of proteomics studies. Hence, this predicted structure can be used for further advance studies and drug designing. Protein interaction analysis shows that CHRNA7 protein also interact with AKT1 protein which regulate neuronal differentiation and development, that signifies the role of CHRNA7 protein in neurological diseases.

Genes encoding subunits of the mitochondrial ribosomal protein complex are distributed between the mitochondrial and nuclear genomes. In Arabidopsis thaliana only seven out of the nearly 70 genes coding for mitochondrial ribosomal subunit proteins are present in the mitochondrial genome. Nevertheless, these genes are co-ordinately expressed. Here, we present the first report of characterization of promoter of a plant mitochondrial ribosomal protein gene AtRps19 (At5g47320), a single copy nuclear gene in A. thaliana. Analysis of transgenic A. thaliana plants carrying seven different AtRps19 upstream fragments linked to the uidA reporter gene revealed that the 879 bp fragment containing the 5′ UTR and the intergenic region is capable of driving gene expression in most of the vegetative tissues. However, inclusion of 447 bp of the upstream gene sequences was essential for obtaining full expression in all tissues including anthers and pollen. Thus, we provide the first experimental proof of overlapping genes in plants. qRT-PCR analysis showed that uidA and native AtRps19 transcript levels were comparable in plants carrying the D4 (−676/+650) construct, indicating that the 1326 bp D4 fragment represents the native promoter of the AtRps19 gene. Comparison of AtRps19 promoter with the widely used CaMV35S promoter showed that AtRps19 promoter is capable of driving gene expression in all tissues including anthers and pollen but was less efficient than the CaMV35S promoter.

Pseudouridylation is a common post‐transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity‐biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post‐transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine‐Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.

Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and well-characterized posttranslational modification known. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) Shewanella-like protein phosphatase 2 (AtSLP2) is a bona fide Ser/Thr protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (AtMIA40), forming a protein complex. Interaction with AtMIA40 is necessary for the phosphatase activity of AtSLP2 and is dependent on the formation of disulfide bridges on AtSLP2. Furthermore, by utilizing atslp2 null mutant, AtSLP2 complemented and AtSLP2 overexpressing plants, we identify a function for the AtSLP2-AtMIA40 complex in negatively regulating gibberellic acid-related processes during seed germination. Results presented here characterize a mitochondrial IMS-localized protein phosphatase identified in photosynthetic eukaryotes as well as a protein phosphatase target of the highly conserved eukaryotic MIA40 IMS oxidoreductase.

BackgroundMembers of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions.MethodsHEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established.ResultsHere, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content.ConclusionsTaken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

Iron–sulfur (Fe-S) clusters (ISCs) are ubiquitous cofactors essential to numerous fundamental cellular processes. Assembly of ISCs and their insertion into apoproteins involves the function of complex cellular machineries that operate in parallel in the mitochondrial and cytosolic/nuclear compartments of mammalian cells. The spectrum of diseases caused by inherited defects in genes that encode the Fe-S assembly proteins has recently expanded to include multiple rare human diseases, which manifest distinctive combinations and severities of global and tissue-specific impairments. In this review, we provide an overview of our understanding of ISC biogenesis in mammalian cells, discuss recent work that has shed light on the molecular interactions that govern ISC assembly, and focus on human diseases caused by failures of the biogenesis pathway.

The Hsp70 family of heat shock proteins plays a critical function in maintaining cellular homeostasis within various subcellular compartments. The human mitochondrial Hsp70 (HSPA9) has been associated with cellular death, senescence, cancer and neurodegenerative diseases, which is the rational for the name mortalin. It is well documented that mortalin, such as other Hsp70s, is prone to self-aggregation, which is related to mitochondria biogenesis failure. Here, we investigated the assembly, structure and function of thermic aggregates/oligomers of recombinant human mortalin and Hsp70-1A (HSPA1A). Summarily, both Hsp70 thermic aggregates have characteristics of supramolecular assemblies. They display characteristic organized structures and partial ATPase activity, despite their nanometric size. Indeed, we observed that the interaction of these aggregates/oligomers with liposomes is similar to monomeric Hsp70s and, finally, they were non-toxic over neuroblastoma cells. These findings revealed that high molecular mass oligomers of mortalin and Hsp70-1A preserved some of the fundamental functions of these proteins.

Current ovarian cancer maintenance therapy is limited by toxicity and no proven impact on overall survival. To study a maintenance strategy targeted at missense mutant p53, we hypothesized that the release of mutant p53 from mortalin inhibition by the SHetA2 drug combined with reactivation of mutant p53 with the PRIMA-1(MET) drug inhibits growth and tumor establishment synergistically in a mutant-p53 dependent manner. The Cancer Genome Atlas (TCGA) data and serous ovarian tumors were evaluated for TP53 and HSPA9/mortalin status. SHetA2 and PRIMA-1(MET) were tested in ovarian cancer cell lines and fallopian tube secretory epithelial cells using isobolograms, fluorescent cytometry, Western blots and ELISAs. Drugs were administered to mice after peritoneal injection of MESOV mutant p53 ovarian cancer cells and prior to tumor establishment, which was evaluated by logistic regression. Fifty-eight percent of TP53 mutations were missense and there were no mortalin mutations in TCGA high-grade serous ovarian cancers. Mortalin levels were sequentially increased in serous benign, borderline and carcinoma tumors. SHetA2 caused p53 nuclear and mitochondrial accumulation in cancer, but not in healthy, cells. Endogenous or exogenous mutant p53 increased SHetA2 resistance. PRIMA-1(MET) decreased this resistance and interacted synergistically with SHetA2 in mutant and wild type p53-expressing cell lines in association with elevated reactive oxygen species/ATP ratios. Tumor-free rates in animals were 0% (controls), 25% (PRIMA1(MET)), 42% (SHetA2) and 67% (combination). SHetA2 (p = 0.004) and PRIMA1(MET) (p = 0.048) functioned additively in preventing tumor development with no observed toxicity. These results justify the development of SHetA2 and PRIMA-1(MET) alone and in combination for ovarian cancer maintenance therapy.

Worldwide, lung cancer is a leading cause of cancer-related deaths and is the most commonly diagnosed form of cancer. A major characteristic of lung cancer is its profound clinical, histological and molecular heterogeneity. This heterogeneity is not only spatial but also temporal thus stressing the need for personalized patient-tailored treatment planning. The current optimal treatment planning is currently based on real-time monitoring of the evolving molecular profiling of the tumour throughout the course of the disease and treatment. In the current work, we will investigate the emerging role that that RPL9 and LIAS could have in carcinogenesis. While the aberrant expression of RPL9 has already been shown to occur in colorectal cancer its role in lung cancer is not yet known. In a similar manner, the role of LIAS, as a metabolism-linked gene, in cancer biology and especially in lung cancer is still unknown. Emerging research reveals both RPL9 and LIAS as interacting partners and apoptosis resistance genes. The aim of this study is to determine the differential expression of the rpl9 and lias genes in both normal lung tissue and lung cancer samples. This was achieved by using in situ hybridization (ISH) and quantitative Real-time PCR (qPCR). Further data on the role played by RPL9 in lung cancer was established through the use of in silico bioinformatic analysis. This was done in order to map biological pathways enriched by the expression of these genes. Both the KEGG pathway and Reactome analysis confirmed the role of these genes in RNA metabolic pathways. Furthermore, RPL9 was shown to play a role in signal transduction, autophagy, and cellular response to stress pathways. The function of these two proteins overlapped with regard to protein metabolism. STRING analysis also demonstrated an interaction between RPL9 and LIAS. Here we propose that the aberrant expression of RPL9 and LIAS may contribute to lung carcinogenesis and can be targeted for molecular therapy.

Mitochondrial ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. Here, we demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function.

We have analyzed the impact of Parkinson's disease (PD)–related genetic variants on splicing using dedicated minigene assays. Out of 14 putative splicing variants in 5 genes (PINK1, [PTEN induced kinase 1]; LRPPRC, [leucine-rich pentatricopeptide repeat containing protein]; TFAM, [mitochondrial transcription factor A]; PARK2, [parkin RBR E3 ubiquitin protein ligase]; and HSPA9, [heat shock protein family A (Hsp70) member 9]), 4 LRPPRC variants, (IVS32−3C>T, IVS35+14C>T, IVS35+15C>T, and IVS9+30A>G) influenced, pre-messenger RNA splicing by modulating the inclusion of the respective exons. In addition, 1-Methyl-4-phenylpyridinium ion–induced splicing changes of endogenous LRPPRC messenger RNA, reproduced the effect of the LRPPRC IVS35+14C>T mutation. Using silencing and overexpression methods, we show that LRPPRC exon 33 splicing is negatively regulated by heterogeneous nuclear ribonucleoprotein A1 both in a minigene and endogenous context. Furthermore, exon 33 exclusion due to PD-associated mutation IVS32−3C>T or heterogeneous nuclear ribonucleoprotein A1 overexpression and exon 35 exclusion due to IVS35+14C>T can be rescued by co-expression of modified U1 small nuclear RNAs, providing a potentially useful therapeutic strategy. Our results indicate for the first time that LRPPRC intronic variants can affect normal splicing of this gene and may influence disease risk in PD and related disorders.

Since Green Fluorescent Protein (GFP) was first successfully expressed in heterologous systems in 1994, many genes encoding other natural autofluorescent proteins (AFPs) have been cloned and subsequently modified by protein engineering to improve their physicochemical properties. Throughout this twenty-two-year period, glycine 67 (Gly67) has been regarded as the only amino acid in the entire protein family that is essential for the formation of the different reported chromophores. In this work, we demonstrate that a synthetic gene encoding LanFP10-A, a natural protein encoded in the genome of the lancelet Branchiostoma floridae containing the G67A mutation, produces a heterologous, functional yellow fluorescent protein when expressed in E. coli. In contrast to LanFP10-A, LanFP6-A, a second GFP-like protein found in the lancelet genome that also contains the natural G67A mutation, was non-fluorescent.

Mitochondria are multifunctional cellular organelles that host many biochemical pathways including oxidative phosphorylation (OXPHOS). Defective mitochondria pose a threat to cellular homeostasis and compensatory responses exist to curtail the source of stress and/or its consequences. The mitochondrial proteome comprises proteins encoded by the nuclear and mitochondrial genomes. Disturbances in protein homeostasis may originate from mistargeting of nuclear encoded mitochondrial proteins. Defective protein import and accumulation of mistargeted proteins leads to stress that triggers translation alterations and proteasomal activation. These cytosolic pathways are complementary to the mitochondrial unfolded protein response (UPRmt) that aims to increase the capacity of protein quality control mechanisms inside mitochondria. They constitute putative targets for interventions aimed at increasing the fitness, stress resistance, and longevity of cells and organisms.

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

Mitochondrial Hsp70 (HSPA9, mtHsp70, mortalin) in conjunction with a complex set of other proteins is involved in the transport of polypeptides across the mitochondrial matrix. This observation allows us to hypothesize that HSPA9 might interact with membranes directly, similarly to other Hsp70s. Thus, we investigated whether human HSPA9 could also get inserted into lipid membranes. Human HSPA9 was incubated with liposomes made of lipids found within the mitochondrial membrane, such as 1', 3'-bis [1, 2-dimyristoyl-sn-glycero-3-phospho]-glycerol (CL), palmitoyl-oleoyl phosphocholine (POPC), palmitoyl-oleoyl phosphoserine (POPS), and palmitoyl-oleoyl phosphoethanolamine (POPE). HSPA9 displayed a predilection for CL and POPS, and low affinity for POPC and POPE, suggesting that the proteins have high specificity for negatively charged phospholipids. Then, liposomes were made with a composition resembling either the outer or inner mitochondrial membrane (OMM or IMM, respectively). We observed that HSPA9 has a higher affinity for IMM than OMM, which is consistent with the higher content of CL in the IMM. A comparison for the incorporation into POPS or CL liposomes by HSPA9 or HSPA1 indicated that both proteins behaved very similarly when exposed to CL liposomes, but differently with POPS liposomes, which was further corroborated by their susceptibility to proteinase K digestion after incorporation into liposomes. The measurement of thermodynamic parameters also showed that the interaction of both proteins with CL and POPS liposomes was different. Overall, our data showed that HSPA9 is prone to interact with membranes resembling the IMM that may be important for its role in the translocation of proteins into the mitochondria.

C. xanthorrhiza and C. zedoaria, anticancer agent widely used in Asia. Previous studies have identified that active compounds from both Curcuma are capable of inhibiting cancer cell growth and inducing apoptosis. However, the roles from its active compound for Mortalin inhibition in carcinogenesis process was unknown. The recent study shows that Mortalin also known as HSPA9/GRP75/mtHSP70 is a highly conserved heat-shock protein 70 family has various roles in carcinogenesis process in multiple ways and very complicated. This study analyzed the potential of active compound from both curcuma as Mortalin inhibitor to control cancer cell growth with the computational study. the results of binding affinity analysis through molecular docking show that some active compounds from both Curcuma have the potential as Mortalin inhibitor with smallest energy and same binding site with commercial Mortalin inhibitors. protein interaction analysis illustrates that Mortalin can interact with various protein including hspd1, timm17a, and hspa4. upregulation is it proteins expression have been identified as a diagnostic marker and poor prognosis factor for cancer. these data indicate that active compounds from both Curcuma have a promising opportunity as anticancer through Mortalin inhibitor mechanism. additional research is needed to validate the in vitro activity of the compounds.

Background: Cisplatin (CDDP), a small molecule platinum-based compound, is an effective anticancer drug used against a wide range of human neoplasms. Long-term clinical use of CDDP is however limited due to the development of drug resistance and the possible incidence of serious side effects including nephrotoxicity and ototoxicity. The mechanisms underlying resistance of cells to CDDP are complex, and among them, the cytoprotective involvement of proteins referred to as Heat Shock Proteins (HSP) seems potentially important. Methods: We searched various electronic databases including PubMed and selected the reports concerning the contribution of HSPs to CDDP resistance of cancer cells and to minimize the CDDP-induced nephrotoxicity and ototoxicity. Results: This critical review of data collected so far summarizes the results on the major HSPs: HSP27/HSPB1, HSP70/HSPA1, HSP90/HSPC and GRP78/HSPA5, because only these have been the subject of the most intense research in the matter discussed here. We also provide relevant information concerning some other HSPs, namely HSPA9/mortalin, HSPA2, HSP110 and DNAJ. A possible role of HSPs in counteracting CDDP-induced neprho- and ototoxicity is mentioned. Conclusions: This review shows that no universal relationship between the levels of expression of HSPs and sensitivity of cancer cells to CDDP can be confirmed. Multiple observations indicate however that such correlation can rather manifest as a molecular or cellular context-dependent phenomenon. Thus, HSPs can be viewed as an important component of the multifactorial, complex response of cancer cells to CDDP. However, to strengthen such a conviction, more extensive studies are needed.

Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses non-canonical mitochondrial protein import machinery. Previously, we characterized the essential translocase of the mitochondrial inner membrane (TIM) consisting of Tim17 in T. brucei. TbTim17 is associated with TbTim62. Here we show that TbTim62, a novel protein, is localized in the mitochondrial inner membrane, and its import into mitochondria depends on TbTim17. Knockdown (KD) of TbTim62 decreased the steady-state levels of TbTim17 post-transcriptionally. Further analysis showed that import of TbTim17 into mitochondria was not inhibited, but its half-life was reduced >4-fold due to TbTim62 KD. Blue-native gel electrophoresis revealed that TbTim62 is present primarily in ∼150-kDa and also in ∼1100-kDa protein complexes, whereas TbTim17 is present in multiple complexes within the range of ∼300 to ∼1100 kDa. TbTim62 KD reduced the levels of both TbTim62 as well as TbTim17 protein complexes. Interestingly, TbTim17 was accumulated as lower molecular mass complexes in TbTim62 KD mitochondria. Furthermore, depletion of TbTim62 hampered the assembly of the ectopically expressed TbTim17–2X-myc into TbTim17 protein complex. Co-immunoprecipitation analysis revealed that association of TbTim17 with mHSP70 was markedly reduced in TbTim62 KD mitochondria. All together our results demonstrate that TbTim62, a unique mitochondrial protein in T. brucei, is required for the formation of a stable TbTim17 protein complex. TbTim62 KD destabilizes this complex, and unassembled TbTim17 degrades. Therefore, TbTim62 acts as a novel regulatory factor to maintain the levels of TIM in T. brucei mitochondria.

The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA(Ile) lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins.

Mitochondrial genome is responsible for multiple human diseases in a maternal inherited pattern, yet phenotypes of patients in a same pedigree frequently vary largely. Genes involving in epigenetic modification, RNA processing, and other biological pathways, rather than "threshold effect" and environmental factors, provide more specific explanation to the aberrant phenotype. Thus, the double hit theory, mutations both in mitochondrial DNA and modifying genes aggravating the symptom, throws new light on mitochondrial dysfunction processes. In addition, mitochondrial retrograde signaling pathway that leads to reconfiguration of cell metabolism to adapt defects in mitochondria may as well play an active role. Here we review selected examples of modifier genes and mitochondrial retrograde signaling in mitochondrial disorders, which refine our understanding and will guide the rational design of clinical therapies.

The function of mitochondria depends on the import of proteins, which are synthesized as precursors on cytosolic ribosomes. The majority of the precursor proteins are sorted into the mitochondrial subcompartments via five distinct routes. Recent studies revealed that molecular cooperation between protein machineries is a central feature of mitochondrial protein biogenesis. First, coupling to various partner proteins affects the substrate specificity of translocases and single translocation steps. Second, there is a substantial cooperation between different protein translocases in the import of specific precursor proteins. Third, protein transport is intimately linked to processing, folding and assembly reactions. Fourth, sorting of precursor proteins is functionally and physically connected to protein machineries, which fulfill central functions for respiration, maintenance of membrane architecture and form contacts to the endoplasmic reticulum. Therefore, we propose that the protein transport systems are part of a complicated protein network for mitochondrial biogenesis.

Sphingomyelin synthase 1 (SMS1) catalyses the biosynthesis of sphingomyelin in eukaryotic cells. We have previously determined the structure of the SGMS1 gene encoding this enzyme and a number of its alternative transcripts. Here, we describe a study of the expression of the full-length SMS1 protein and the sum of the alternative transcripts encoding this protein in human tissues. The SMS1 protein and mRNA levels in tissues differed significantly and were not correlated, implying the active post-transcriptional regulation of SMS1 protein expression. The putative truncated isoforms of the SMS1 protein, which are encoded by a number of alternative transcripts, were not detected by immunoblotting and thus may be absent or present in only small amounts.

Precursor proteins containing mitochondrial peptide signals are cleaved after import by a mitochondrial processing peptidase. In yeast (Saccharomyces cerevisiae) and human (Homo sapiens), intermediate cleavage peptidase55 (ICP55) plays a role in stabilizing mitochondrial proteins by the removal of single amino acids from mitochondrial processing peptidase-processed proteins. We have investigated the role of a metallopeptidase (At1g09300) from Arabidopsis (Arabidopsis thaliana) that has sequence similarity to yeast ICP55. We identified this protein in mitochondria by mass spectrometry and have studied its function in a transfer DNA insertion line (icp55). Monitoring of amino-terminal peptides showed that Arabidopsis ICP55 was responsible for the removal of single amino acids, and its action explained the -3 arginine processing motif of a number of mitochondrial proteins. ICP55 also removed single amino acids from mitochondrial proteins known to be cleaved at nonconserved arginine sites, a subset of mitochondrial proteins specific to plants. Faster mitochondrial protein degradation rates not only for ICP55 cleaved protein but also for some non-ICP55 cleaved proteins were observed in Arabidopsis mitochondrial samples isolated from icp55 than from the wild type, indicating that a complicated protease degradation network has been affected. The lower protein stability of isolated mitochondria and the lack of processing of target proteins in icp55 were complemented by transformation with the full-length ICP55. Analysis of in vitro degradation rates and protein turnover rates in vivo of specific proteins indicated that serine hydroxymethyltransferase was affected in icp55. The maturation of serine hydroxymethyltransferase by ICP55 is unusual, as it involves breaking an amino-terminal diserine that is not known as an ICP55 substrate in other organisms and that is typically considered a sequence that stabilizes rather than destabilizes a protein.

The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein–protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to endosomal sorting complex required for transport-I components. Disruption of any of these interactions results in abscission failure, by disrupting the proper recruitment of CEP55, and subsequently, of endosomal sorting complex required for transport-I, to the midbody. Our data suggest that myotubularin-related protein 3 and myotubularin-related protein 4 may act as a bridge between CEP55 and polo-like kinase 1, ensuring proper CEP55 phosphorylation and regulating CEP55 recruitment to the midbody. This work provides a novel role for myotubularin-related protein 3/4 heterodimers, and highlights the temporal and spatial complexity of the regulation of cytokinesis.

Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.

International audience; GALIG, an internal gene to the human galectin-3 gene, encodes two distinct proteins, Mitogaligin and Cytogaligin through translation of a unique mRNA in two overlapping alternative reading frames. When overexpressed GALIG induces apoptosis. In cultured cells, Mitogaligin destabilizes mitochondria membranes through interaction with cardiolipin. Little is known regarding the role of Cytogaligin. This protein displays multiple subcellular localizations; cytosol, nucleus, and mitochondria. We illustrate here that Cytogaligin is also secreted in the extracellular medium. Cytogaligin is shown to interact with α-Synuclein, the major component of Lewy bodies in Parkinson's disease. Overexpression of Cytogaligin reduces α-Synuclein dimerization raising a possible role in the evolution of α-Synuclein aggregation, a key molecular event underlying the pathogenesis of Parkinson's disease.