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Mutations in GBA1, the gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher’s disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employ global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration.

Although the best-known spinocerebellar ataxias (SCAs) are triplet repeat diseases, many SCAs are not caused by repeat expansions. The rarity of individual non-expansion SCAs, however, has made it difficult to discern genotype-phenotype correlations. We therefore screened individuals who had been found to bear variants in a non-expansion SCA-associated gene through genetic testing, and after we eliminated genetic groups that had fewer than 30 subjects, there were 756 subjects bearing single-nucleotide variants or deletions in one of seven genes: CACNA1A (239 subjects), PRKCG (175), AFG3L2 (101), ITPR1 (91), STUB1 (77), SPTBN2 (39), or KCNC3 (34). We compared age at onset, disease features, and progression by gene and variant. There were no features that reliably distinguished one of these SCAs from another, and several genes-CACNA1A, ITPR1, SPTBN2, and KCNC3-were associated with both adult-onset and infantile-onset forms of disease, which also differed in presentation. Nevertheless, progression was overall very slow, and STUB1-associated disease was the fastest. Several variants in CACNA1A showed particularly wide ranges in age at onset: one variant produced anything from infantile developmental delay to ataxia onset at 64 years of age within the same family. For CACNA1A, ITPR1, and SPTBN2, the type of variant and charge change on the protein greatly affected the phenotype, defying pathogenicity prediction algorithms. Even with next-generation sequencing, accurate diagnosis requires dialogue between the clinician and the geneticist.

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. Several lines of evidence suggest that ClpC1P1P2 similarly recognizes phosphoarginine-bearing proteins, but the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate of Mtb. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on a diverse collection of targets within the Msm proteome. Physicochemical and functional characterization of targets suggest that arginine phosphorylation is applied in a sequence-independent manner as part of a proteome-wide quality control pathway. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/438432v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@607043org.highwire.dtl.DTLVardef@16a4614org.highwire.dtl.DTLVardef@1efaf5org.highwire.dtl.DTLVardef@1ee2247_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mitochondrial i-AAA proteinase Yme1 is a multifunctional protein that plays important roles in maintaining mitochondrial protein homeostasis and regulating biogenesis and function of mitochondrial proteins. However, due to the complex interplay of mitochondria and the multifunctional nature of Yme1, how Yme1 affects mitochondrial function and protein homeostasis is still poorly understood. In this study, we investigated how YME1 deletion affects yeast Saccharomyces cerevisiae growth, chronological life span, mitochondrial protein homeostasis and function, with a focus on the mitochondrial oxidative phosphorylation (OXPHOS) complexes. Our results show that whilst the YME1 deleted cells grow poorly under respiratory conditions, they grow similar to wild-type yeast under fermentative conditions. However, the chronological life span is impaired, indicating that Yme1 plays a key role in longevity. Using highly enriched mitochondrial extract and proteomic analysis, we show that the abundances of many mitochondrial proteins are altered by YME1 deletion. Several components of the respiratory chain complexes II, III, IV and V were significantly decreased, suggesting that Yme1 plays an important role in maintaining the level and function of complexes II-V. This result was confirmed using blue native-PAGE and in-solution-based enzyme activity assays. Taken together, this study shows that Yme1 plays an important role in the chronological life span and mitochondrial protein homeostasis and has deciphered its function in maintaining the activity of mitochondrial OXPHOS complexes.

Objectives Clinical diagnostics in adults with hereditary neurological diseases is complicated by clinical and genetic heterogeneity, as well as lifestyle effects. Here, we evaluate the effectiveness of exome sequencing and clinical costs in our difficult-to-diagnose adult patient cohort. Additionally, we expand the phenotypic and genetic spectrum of hereditary neurological disorders in Finland. Methods We performed clinical exome sequencing (CES) to 100 adult patients from Finland with neurological symptoms of suspected genetic cause. The patients were classified as myopathy (n = 57), peripheral neuropathy (n = 16), ataxia (n = 15), spastic paraplegia (n = 4), Parkinsonism (n = 3), and mixed (n = 5). In addition, we gathered the costs of prior diagnostic work-up to retrospectively assess the cost-effectiveness of CES as a first-line diagnostic tool. Results The overall diagnostic yield of CES was 27%. Pathogenic variants were found for 14 patients (in genes ANO5, CHCHD10, CLCN1, DES, DOK7, FKBP14, POLG, PYROXD1, SCN4A, TUBB3, and TTN) and likely pathogenic previously undescribed variants for 13 patients (in genes ABCD1, AFG3L2, ATL1, CACNA1A, COL6A1, DYSF, IRF2BPL, KCNA1, MT-ATP6, SAMD9L, SGCB, and TPM2). Age of onset below 40 years increased the probability of finding a genetic cause. Our cost evaluation of prior diagnostic work-up suggested that early CES would be cost-effective in this patient group, in which diagnostic costs increase linearly with prolonged investigations. Conclusions Based on our results, CES is a cost-effective, powerful first-line diagnostic tool in establishing the molecular diagnosis in adult neurological patients with variable symptoms. Importantly, CES can markedly shorten the diagnostic odysseys of about one third of patients.

Mitochondrial unfolded protein response plays an important role in the occurrence and development of breast cancer. However, the role of mitochondrial unfolded protein response (UPRmt) in the sensitivity of breast cancer to cisplatin chemotherapy has not yet been cleared. The purpose of this study is to explore the role of mitochondrial unfolded protein response in breast cancer sensitivity to cisplatin. In this study, qRT-PCR, Western blotting, Immunofluorescence, CCK-8, Colony formation, Transwell assay and TUNEL staining assay were used to confirm the role of UPRmt in breast cancer cells treated with cisplatin. Cisplatin increased the levels of UPRmt including CLPP, HSP60, LONP1 in MCF7 and MDA-MB-231 cells. UPRmt inducer Nicotinamide ribose (NR) could promote the proliferation and invasion of breast cancer cells treated with cisplatin. Importantly, SIRT3 was discovered to increase UPRmt in breast cancer cells and silencing of SIRT3 could inhibit the effect of NR in breast cancer. UPRmt regulated by SIRT3 could protect breast cancer cell from cisplatin. Controlling SIRT3-induced UPR may be a potential therapeutic target to increase the sensitivity of breast cancer chemotherapy.

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as

Osteoarthritis (OA) is an age-related disorder and an important cause of disability that is characterized by a senescence-associated secretory phenotype and matrix degradation leading to a gradual loss of articular cartilage integrity. Mitochondria, as widespread organelles, are involved in regulation of complex biological processes such as energy synthesis and cell metabolism, which also have bidirectional communication with the nucleus to help maintain cellular homeostasis and regulate adaptation to a broad range of stressors. In light of the evidence that OA is strongly associated with mitochondrial dysfunction. In addition, mitochondria are considered to be the culprits of cell senescence, and mitochondrial function changes during ageing are considered to have a controlling role in cell fate. Mitochondrial dysfunction is also observed in age-related OA, however, the internal mechanism by which mitochondrial function changes with ageing to lead to the development of OA has not been elucidated. In this study, we found that the expression of Lon protease 1 (LONP1), a mitochondrial protease, was decreased in human OA cartilage and in ageing rat chondrocytes. Furthermore, LONP1 knockdown accelerated the progression and severity of osteoarthritis, which was associated with aspects of mitochondrial dysfunction including oxidative stress, metabolic changes and mitophagy, leading to downstream MAPK pathway activation. Antioxidant therapy with resveratrol suppressed oxidative stress and MAPK pathway activation induced by LONP1 knockdown to mitigate OA progression. Therefore, our findings demonstrate that LONP1 is a central regulator of mitochondrial function in chondrocytes and reveal that downregulation of LONP1 with ageing contributes to osteoarthritis.

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress.

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease., The human mitochondrial protease LONP1 is an AAA+ ATP-dependent quality control protease. Here, the authors present the cryo-EM structures of human LONP1 in three distinct states and provide insights into the mechanism and regulation of this important protease.

Little is known about Nima‐related kinase (NEKs), a widely conserved family of kinases that have key roles in cell‐cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase‐3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1‐TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1., mtDNA mutations have been increasingly observed in human cancers. Cumulative mtDNA damage declines cell and tissue function and underpins the aging process. Protein kinases selectively modify proteins and a plethora of signaling pathways, leading to a specific physiological responses. Here, we demonstrate how NEK5 kinase can contribute to the maintenance and repair of mtDNA.

Background Although the typical inheritance of spastic paraplegia 7 is recessive, several reports have suggested that SPG7 variants may also cause autosomal dominant hereditary spastic paraplegia (HSP). Objectives We aimed to conduct an exome-wide genetic analysis on a large Canadian cohort of HSP patients and controls to examine the association of SPG7 and HSP. Methods We analyzed 585 HSP patients from 372 families and 1175 controls, including 580 unrelated individuals. Whole-exome sequencing was performed on 400 HSP patients (291 index cases) and all 1175 controls. Results The frequency of heterozygous pathogenic/likely pathogenic SPG7 variants (4.8%) among unrelated HSP patients was higher than among unrelated controls (1.7%; OR 2.88, 95% CI 1.24-6.66, P = 0.009). The heterozygous SPG7 p.(Ala510Val) variant was found in 3.7% of index patients versus 0.85% in unrelated controls (OR 4.42, 95% CI 1.49-13.07, P = 0.005). Similar results were obtained after including only genetically-undiagnosed patients. We identified four heterozygous SPG7 variant carriers with an additional pathogenic variant in known HSP genes, compared to zero in controls (OR 19.58, 95% CI 1.05-365.13, P = 0.0031), indicating potential digenic inheritance. We further identified four families with heterozygous variants in SPG7 and SPG7-interacting genes (CACNA1A, AFG3L2, and MORC2). Of these, there is especially compelling evidence for epistasis between SPG7 and AFG3L2. The p.(Ile705Thr) variant in AFG3L2 is located at the interface between hexamer subunits, in a hotspot of mutations associated with spinocerebellar ataxia type 28 that affect its proteolytic function. Conclusions Our results provide evidence for complex inheritance in SPG7-associated HSP, which may include recessive and possibly dominant and digenic/epistasis forms of inheritance. © 2021 International Parkinson and Movement Disorder Society.

The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the β2-β3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and in vivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation. Ministry of Education (MOE) Published version This work was supported by a Tier II grant MOE2019-T2-2-099 and a Tier 1 grant RG108/20 from the Ministry of Education (MOE) of Singapore (Y.-G.G.).

Thylakoid FtsH complex participates in PSII repair cycle during high light-induced photoinhibition. The Arabidopsis yellow variegated2 (var2) mutants are defective in the VAR2/AtFtsH2 subunit of thylakoid FtsH complex. Taking advantage of the var2 leaf variegation phenotype, dissections of genetic enhancer loci have yielded novel paradigms in understanding functions of thylakoid FtsH complex. Here, we report the isolation of a new var2 enhancer, enhancer of variegation2-1 (evr2-1). We confirmed that EVR2 encodes a chloroplast protein that was known as BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1), or CHLOROPHYLL BIOSYNTHETIC DEFECT 1 (CBD1). We showed that EVR2/BCM1/CBD1 was involved in the oligomerization of photosystem I complexes. Genetic assays indicated that general defects in chlorophyll biosynthesis and the accumulation of photosynthetic complexes do not necessarily enhance var2 leaf variegation. In addition, we found that VAR2/AtFtsH2 is required for the accumulation of photosynthetic proteins during de-etiolation. Moreover, we identified PSII core proteins D1 and PsbC as potential EVR2-associated proteins using Co-IP/MS. Furthermore, the accumulation of D1 protein was greatly compromised in the var2-5 evr2-1 double mutant during de-etiolation. Together, our findings reveal a functional link between VAR2/AtFtsH2 and EVR2/BCM1/CBD1 in regulating chloroplast development and the accumulation of PSII reaction centre D1 protein during de-etiolation.

Saudi Genome program is a revolutionary nationwide transformation initiative of Saudi Vision 2030. The program goals are to recognize and reduce the incidence of genetic diseases in the Kingdom of Saudi Arabia (KSA). Accordingly, the program will establish the foundation for personalized and genomic medicine in the KSA. Epilepsy has a high prevalence in KSA reaching around 6.54 of 1000 individuals with a subsequent massive financial burden. One of the main risk factors for this high prevalence and associated with increased risk of epilepsy development is consanguinity marriage, which is traditional in KSA. In this review, we executed a comprehensive state-of-art literature review regarding epilepsy genetics to offer a perception into the genes associated with epilepsy recognized in Saudi epileptic patients. Several genes' mutations were incorporated in this review including AFG3L2, ASPM, ATN1, ATP1A2, BMP5, CCDC88A, C12orf57, DNAJA1, EML1, ERLIN2, FRRS1L, GABRG3, NRXN3, MDH1, KCNJ10, KCNMA1, KCNT1, KIAA0226, OPHN1, PCCA, PCCB, PEX, PGAP2, PI4K2A, PODXL, PRICKLE1, PNKP, RELN, SCN2A, SCN1B, SLC2A1, SLC19A3, SLC25, SIAH1, SYNJ1, SZT2, TBCK, TMX2, TSC1, TSC2, TSEN, WDR45B, WWOX, UBR, UGDH, and YIF1B. For each of these genes, we tried to explain a little about the gene associated proteins and their roles in epilepsy development.

The mitochondrial matrix AAA+ Lon protease (LONP1) degrades misfolded or unassembled proteins, which play a pivotal role in mitochondrial quality control. During heart development, a metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation takes place, which relies strongly on functional mitochondria. However, the relationship between the mitochondrial quality control machinery and metabolic shifts is elusive. Here, we interfered with mitochondrial quality control by inactivating Lonp1 in murine embryonic cardiac tissue, resulting in severely impaired heart development, leading to embryonic lethality. Mitochondrial swelling, cristae loss and abnormal protein aggregates were evident in the mitochondria of Lonp1-deficient cardiomyocytes. Accordingly, the p-eIF2α-ATF4 pathway was triggered, and nuclear translocation of ATF4 was observed. We further demonstrated that ATF4 regulates the expression of Tfam negatively while promoting that of Glut1, which was responsible for the disruption of the metabolic shift to oxidative phosphorylation. In addition, elevated levels of reactive oxygen species were observed in Lonp1-deficient cardiomyocytes. This study revealed that LONP1 safeguards metabolic shifts in the developing heart by controlling mitochondrial protein quality, suggesting that disrupted mitochondrial quality control may cause prenatal cardiomyopathy.

Monogenic forms of cerebral small vessel disease (CSVD) can be caused by both variants in nuclear DNA and mitochondrial DNA (mtDNA). Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is known to have a phenotype similar to Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), and can be caused by variants in the mitochondrial genome and in several nuclear-encoded mitochondrial protein (NEMP) genes. The aim of this study was to screen for variants in the mitochondrial genome and NEMP genes in a NOTCH3-negative CADASIL cohort, to identify a potential link between mitochondrial dysfunction and CSVD pathology. Whole exome sequencing was performed for 50 patients with CADASIL-like symptomology on the Ion Torrent system. Mitochondrial sequencing was performed using an in-house designed protocol with sequencing run on the Ion GeneStudio S5 Plus (S5 +). NEMP genes and mitochondrial sequencing data were examined for rare (MAF POLG, MTO1, LONP1, NDUFAF6, NDUFB3, and TCIRG1 were thought to play a potential role in CSVD pathology in this cohort. Overall, the exploration of the mitochondrial genome identified a potential role for mitochondrial related proteins and mtDNA variants contributing to CSVD pathologies.

Objective Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, with a prevalence of 1:12,000 to 1:25,000. OPA1 mutations are found in 70% of DOA patients, with a significant number remaining undiagnosed. Methods We screened 286 index cases presenting optic atrophy, negative for OPA1 mutations, by targeted next generation sequencing or whole exome sequencing. Pathogenicity and molecular mechanisms of the identified variants were studied in yeast and patient-derived fibroblasts. Results Twelve cases (4%) were found to carry novel variants in AFG3L2, a gene that has been associated with autosomal dominant spinocerebellar ataxia 28 (SCA28). Half of cases were familial with a dominant inheritance, whereas the others were sporadic, including de novo mutations. Biallelic mutations were found in 3 probands with severe syndromic optic neuropathy, acting as recessive or phenotype-modifier variants. All the DOA-associated AFG3L2 mutations were clustered in the ATPase domain, whereas SCA28-associated mutations mostly affect the proteolytic domain. The pathogenic role of DOA-associated AFG3L2 mutations was confirmed in yeast, unraveling a mechanism distinct from that of SCA28-associated AFG3L2 mutations. Patients' fibroblasts showed abnormal OPA1 processing, with accumulation of the fission-inducing short forms leading to mitochondrial network fragmentation, not observed in SCA28 patients' cells. Interpretation This study demonstrates that mutations in AFG3L2 are a relevant cause of optic neuropathy, broadening the spectrum of clinical manifestations and genetic mechanisms associated with AFG3L2 mutations, and underscores the pivotal role of OPA1 and its processing in the pathogenesis of DOA. ANN NEUROL 2020 ANN NEUROL 2020;88:18-32.

Lon proteases, members of the AAA(+) superfamily of enzymes, are key components of the protein quality control system in bacterial cells, as well as in the mitochondria and other specialized organelles of higher organisms. These enzymes have been subject of extensive biochemical and structural investigations, resulting in 72 crystal and solution structures, including structures of the individual domains, multi-domain constructs, and full-length proteins. However, interpretation of the latter structures still leaves some questions unanswered. Based on their amino acid sequence and details of their structure, Lon proteases can be divided into at least three subfamilies, designated as LonA, LonB, and LonC. Protomers of all Lons are single-chain polypeptides and contain at least two functional domains, ATPase and protease. The LonA enzymes additionally include a large N-terminal region, and different Lons may also include non-conserved inserts in the principal domains. These ATP-dependent proteases function as homohexamers, in which unfolded substrates are translocated to a large central chamber where they undergo proteolysis by a processive mechanism. X-ray crystal structures provided high-resolution models which verified that Lons are hydrolases with the rare Ser-Lys catalytic dyad. Full-length LonA enzymes have been investigated by cryo-electron microscopy (cryo-EM), providing description of the functional enzyme at different stages of the catalytic cycle, indicating extensive flexibility of their N-terminal domains, and revealing insights into the substrate translocation mechanism. Structural studies of Lon proteases provide an interesting case for symbiosis of X-ray crystallography and cryo-EM, currently the two principal techniques for determination of macromolecular structures.

Mitochondrial proteolysis is an evolutionarily conserved quality-control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a role in controlling mitochondrial function as well as skeletal muscle mass and strength in response to muscle disuse. In humans and mice, disuse-related muscle loss is associated with decreased mitochondrial LONP1 protein. Skeletal muscle-specific ablation of LONP1 in mice resulted in impaired mitochondrial protein turnover, leading to mitochondrial dysfunction. This caused reduced muscle fiber size and strength. Mechanistically, aberrant accumulation of mitochondrial-retained protein in muscle upon loss of LONP1 induces the activation of autophagy-lysosome degradation program of muscle loss. Overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known protein degraded by LONP1, in skeletal muscle induces mitochondrial dysfunction, autophagy activation, and cause muscle loss and weakness. Thus, these findings reveal a role of LONP1-dependent mitochondrial protein quality-control in safeguarding mitochondrial function and preserving skeletal muscle mass and strength, and unravel a link between mitochondrial protein quality and muscle mass maintenance during muscle disuse.

Purpose. Skin cutaneous melanoma (SKCM) is one of the most malignant and aggressive cancers with poor prognosis due to its rapid progression towards metastasis. Thus, finding clinically relevant biomarkers for early diagnosis, prognosis, and therapy prediction is essential. This study focused on the identification of SLC25A13 as a novel biomarker for SKCM and is aimed at investigating the biological functions of solute carrier family 25 member 13 (SLC25A13) in the development of SKCM. Methods. GEPIA was used to analyze the diagnostic and prognostic values of SLC25A13 in SKCM using the TCGA dataset. PrognoScan was used to validate the prognostic value of SLC25A13 and its coexpressed genes in SKCM. TISIDB was established to reveal the relationship between the expression of SLC25A13 and immune infiltration in SKCM. The protein expression of SLC25A13 in SKCM was evaluated by the Human Protein Atlas. The signaling pathways and biological functions of SLC25A13 in SKCM were analyzed by LinkOmics. Metascape was applied to analyze the functional enrichment analysis of SLC25A13. Protein-protein interaction analysis of SLC25A13 was performed by GeneMANIA. Results. The mRNA and protein levels of SLC25A13 in the SKCM were much higher than those in the normal tissue. Furthermore, the overexpression of SLC25A13 predicts worse outcomes of SKCM patients. Moreover, the SLC25A13 expression was negatively correlated with the immune infiltration level of SKCM. The overexpression of SLC25A13 coexpressed genes, such as ACLY and AFG3L2, and SCL25A13 interacting genes also predicted the unfavorable prognosis of SKCM patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of SLC25A13 coexpressed genes showed that these genes are enriched in ATPase activity, cell cycle, mTOR, and VEGFA-VEGFR2 signaling pathways, which were relevant to tumor development and angiogenesis. Gene set enrichment analysis (GSEA) demonstrated that the SLC25A13 expression was related to infiltrating immune cells in SKCM. Conclusion. Our findings revealed that SLC25A13 might be a potential prognostic and therapeutic biomarker for SKCM.

International audience; In Bacillus subtilis, sporulation is a sequential and highly regulated process. Phosphorylation events by histidine kinases are key points in the phosphorelay that initiates sporulation, but serine/threonine protein kinases also play important auxiliary roles in this regulation. PrkA has been proposed to be a serine protein kinase expressed during the initiation of sporulation and involved in this differentiation process. Additionally, the role of PrkA in sporulation has been previously proposed to be mediated via the transition phase regulator ScoC, which in turn regulates the transcriptional factor σK and its regulon. However, the kinase activity of PrkA has not been clearly demonstrated, and neither its autophosphorylation nor phosphorylated substrates have been unambiguously established in B. subtilis. We demonstrated here that PrkA regulation of ScoC is likely indirect. Following bioinformatic homology searches, we revealed sequence similarities of PrkA with the AAA+ ATP-dependent Lon protease family. Here, we showed that PrkA is indeed able to hydrolyze α-casein, an exogenous substrate of Lon proteases, in an ATP-dependent manner. We also showed that this ATP-dependent protease activity is essential for PrkA function in sporulation, since mutation in the Walker A motif leads to a sporulation defect. Furthermore, we found that PrkA protease activity is tightly regulated by phosphorylation events involving one of the Ser/Thr protein kinases of B. subtilis, PrkC. Taken together, our results clarify the key role of PrkA in the complex process of B. subtilis sporulation.

Chloroplast development and photosynthesis require the proper assembly and turnover of photosynthetic protein complexes. Chloroplasts harbor a repertoire of proteases to facilitate proteostasis and development. We have previously used an Arabidopsis leaf variegation mutant, yellow variegated2 (var2), defective in thylakoid FtsH protease complexes, as a tool to dissect the genetic regulation of chloroplast development. Here, we report a new genetic enhancer mutant of var2, enhancer of variegation3–1 (evr3–1). We confirm that EVR3 encodes a chloroplast metalloprotease, reported previously as ethylene-dependent gravitropism-deficient and yellow-green1 (EGY1)/ammonium overly sensitive1 (AMOS1). We observed that mutations in EVR3/EGY1/AMOS1 cause more severe leaf variegation in var2–5 and synthetic lethality in var2–4. Using a modified blue-native PAGE system, we reveal abnormal accumulations of photosystem I, photosystem II, and light-harvesting antenna complexes in EVR3/EGY1/AMOS1 mutants. Moreover, we discover distinct roles of VAR2 and EVR3/EGY1/AMOS1 in the turnover of photosystem II reaction center under high light stress. In summary, our findings indicate that two chloroplast metalloproteases, VAR2/AtFtsH2 and EVR3/EGY1/AMOS1, function coordinately to regulate chloroplast development and reveal new roles of EVR3/EGY1/AMOS1 in regulating chloroplast proteostasis in Arabidopsis.

Rationale: Spinocerebellar ataxia (SCA) is a common neurogenetic disease that mainly manifests as ataxia of posture, gait, and limbs, cerebellar dysarthria, and cerebellar and supranuclear eye movement disorders. SCA has been found to include many subtypes, which are mainly mapped to 2 genetic patterns: autosomal dominant cerebellar ataxia and autosomal recessive cerebellar ataxia. Molecular genetic diagnosis functions as a necessity in its clinical diagnosis and treatment. In preliminary clinical work, we identified a family of SCA28 with rare gene mutation. Patient concerns: There are 5 patients in this family. The proband is a 32 year-old male, he mainly manifest unsteady steps for more than 7 months. The daughter of his younger maternal uncle gradually had unsteady steps and unclear speech for 5 years. The proband's mother, uncle and grandfather had similar symptoms, but they all died. Diagnosis: After Brain magnetic resonance imaging, whole exome sequencing and Sanger validation, the patients presented a c.1852A > G missense mutation in the exon region of AFG3L2 gene. The other family members revealed no AFG3L2 mutations. SCA28 is the one uniquely caused by a pathogenic variation in the mitochondrial protein AFG3L2. Combined with the clinical manifestations, auxiliary examinations and sequencing results of the patients (III-3 and III-5), the diagnosis of SCA28 was suspected. Interventions: The patients did not receive any drug treatment and the proband receive rehabilitation treatment. Outcomes: The symptoms of ataxia were still progressively aggravated. Lessons: Molecular genetic diagnosis is necessary for ataxia. We here report the case and review the literature.

Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher expression of LonP1 has been associated with higher tumour aggressiveness. Besides the full-length isoform (ISO1), we identified two other isoforms of Lonp1 in humans, resulting from alternative splicing: Isoform-2 (ISO2) lacking aa 42-105 and isoform-3 (ISO3) lacking aa 1-196. An inspection of the public database TSVdb showed that ISO1 was upregulated in lung, bladder, prostate, and breast cancer, ISO2 in all the cancers analysed (including rectum, colon, cervical, bladder, prostate, breast, head, and neck), ISO3 did not show significant changes between cancer and normal tissue. We overexpressed ISO1, ISO2, and ISO3 in SW620 cells and found that the ISO1 isoform was exclusively mitochondrial, ISO2 was present in the organelle and in the cytoplasm, and ISO3 was exclusively cytoplasmatic. The overexpression of ISO1 and, at a letter extent, of ISO2 enhanced basal, ATP-linked, and maximal respiration without altering the mitochondria number or network, mtDNA amount. or mitochondrial dynamics. A higher extracellular acidification rate was observed in ISO1 and ISO2, overexpressing cells, suggesting an increase in glycolysis. Cells overexpressing the different isoforms did not show a difference in the proliferation rate but showed a great increase in anchorage-independent growth. ISO1 and ISO2, but not ISO3, determined an upregulation of EMT-related proteins, which appeared unrelated to higher mitochondrial ROS production, nor due to the activation of the MEK ERK pathway, but rather to global metabolic reprogramming of cells.

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1's enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

AAA+ proteases regulate numerous physiological and cellular processes through tightly regulated proteolytic cleavage of protein substrates driven by ATP hydrolysis. FtsH is the only known family of membrane-anchored AAA+ proteases essential for membrane protein quality control. Although a spiral staircase rotation mechanism for substrate translocation across the FtsH pore has been proposed, the detailed conformational changes among various states have not been clear due to absence of FtsH structures in these states. We report here the cryo-EM structure for Thermotoga maritima FtsH (TmFtsH) in a fully ADP-bound symmetric state. Comparisons of the ADP-state structure with its apo-state and a substrate-engaged yeast YME1 structure show conformational changes in the ATPase domains, rather than the protease domains. A reconstruction of the full-length TmFtsH provides structural insights for the dynamic transmembrane and the periplasmic domains. Our structural analyses expand the understanding of conformational switches between different nucleotide states in ATP hydrolysis by FtsH.