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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 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.

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.

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.

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.

Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. Here we show that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo. These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target.

While heterozygous mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia 28 (SCA28), homozygous mutations in the same gene can cause spastic ataxia 5 (SPAX5). AFG3L2 encodes a mitochondrial ATP-dependent metalloprotease. We here report a SCA28 patient with biallelic AFG3L2 variants and his heterozygous mother. The patient and his mother underwent a detailed neurological examination and fibroblast lines were established. The effect of the two missense variants on mitochondria was assessed by form factor analysis and quantification of mitochondrial proteins (TOMM70, complex V). The 39-year-old index patient presented with a slowly progressive cerebellar gait disorder for 19 years, bilateral ptosis, and dysarthria. A cranial MRI showed mild cerebellar atrophy. He carried two compound-heterozygous, rare, missense variants (c.1847A>G [p.Y616C], c.2167G>A [p.V723M]) in AFG3L2, while his mother was heterozygous for the first change that had previously been described in SPAX5. Altered mitochondrial morphology and interconnectivity, together with reduced protein levels of TOMM70 and complex V (ATPase), suggest mitochondrial structural defects in the patient’s fibroblasts. No significant abnormalities were found in his mother’s fibroblast cultures albeit all measurements were slightly below the control level. We here present a SCA28 patient with compound-heterozygous AFG3L2 variants and demonstrate mitochondrial abnormalities in skin fibroblast cultures from this patient. Thus, AFG3L2 variants should be considered in both slowly progressive ataxias and phenotypes with clinical features reminiscent of mitochondrial disease. Of note, ptosis was present in both mutation carriers and may serve as a red flag in the diagnosis of SCA28.

Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation. Abbreviations: ATG: autophagy-related; ATI: ATG8-interacting protein; Col-0: Columbia-0; DSK2: dominant suppressor of KAR2; EMS: ethyl methanesulfonate; GFP: green fluorescent protein; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; ICL: isocitrate lyase; MLS: malate synthase; NBR1: Next to BRCA1 gene 1; PEX: peroxin; PMDH: peroxisomal malate dehydrogenase; PTS: peroxisomal targeting signal; thiolase: 3-ketoacyl-CoA thiolase; UBA: ubiquitin-associated; WT: wild type

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.

The AFG3L2 gene encodes AFG3-like protein 2, which is a subunit of human mitochondrial ATPases associated with various cellular protease activities (m-AAA). The clinical spectrum of AFG3L2 mutations is broad. Dominant AFG3L2 mutations can cause autosomal dominant spinocerebellar ataxia type 28 (SCA28), whereas biallelic AFG3L2 mutations may lead to spastic ataxia 5 (SPAX5). However, the role of AFG3L2 mutations in autosomal recessive spinocerebellar ataxia (SCAR) remains elusive. The aim of this study is to delineate the clinical features and spectrum of AFG3L2 mutations in a Taiwanese cohort with cerebellar ataxia. Mutational analyses of AFG3L2 were carried out by targeted resequencing in a cohort of 133 unrelated patients with molecularly undetermined cerebellar ataxia. We identified one single patient carrying compound heterozygous mutations in AFG3L2, p.[R632*];[V723M] (c.[1894C > T];[2167G > A]). The patient has suffered from apparently sporadic and slowly progressive cerebellar ataxia, ptosis, and ophthalmoparesis since age 55 years. These findings expand the clinical spectrum of AFG3L2 mutations and suggest a new subtype of late-onset SCAR caused by biallelic AFG3L2 mutations.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies., Graphical Abstract Graphical AbstractThe A449T mutation in mouse POLγA, corresponding to the common A467T in patients, impairs the interaction between POLγA and B subunits, making POLγA amenable to degradation by LONP1 protease.

Most mitochondrial precursor polypeptides are imported from the cytosol into the mitochondrion, where they must efficiently undergo folding. Mitochondrial precursors are imported as unfolded polypeptides. For proteins of the mitochondrial matrix and inner membrane, two separate chaperone systems, HSP60 and mitochondrial HSP70 (mtHSP70), facilitate protein folding. We show that LONP1, an AAA+ protease of the mitochondrial matrix, works with the mtHSP70 chaperone system to promote mitochondrial protein folding. Inhibition of LONP1 results in aggregation of a protein subset similar to that caused by knockdown of DNAJA3, a co-chaperone of mtHSP70. LONP1 is required for DNAJA3 and mtHSP70 solubility, and its ATPase, but not its protease activity, is required for this function. In vitro, LONP1 shows an intrinsic chaperone-like activity and collaborates with mtHSP70 to stabilize a folding intermediate of OXA1L. Our results identify LONP1 as a critical factor in the mtHSP70 folding pathway and demonstrate its proposed chaperone activity., Most mitochondrial proteins are imported from the cytosol and must fold in the mitochondria. Here, the authors show that the mitochondrial protease LONP1 plays a critical role in the mtHSP70 chaperone system independently of its protease activity.

The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development., Modulation of metalloprotease FtsH12 abundance in Arabidopsis results in altered composition of the plastid import machinery, which in turn affects the development of functional chloroplasts.

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of &Delta, lapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of &Delta, lapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.

ATP-dependent Lon proteases function in bacterial pathogenesis by regulating the expression of the Type III secretion system; however, little is known about how Lon proteases regulate fungal pathogenesis. We previously investigated Lon-binding proteins involved in fungal pathogenesis that interact with PrePL, the smallest Magnaporthe oryzae Lon-binding protein. Here, we show that Lon cleaves PrePL and produces Pc, an extracellular 11-kDa isoform with catalase and peroxidase activity. The ΔPrePL loss-of-function strain showed stronger sporulation and accelerated disease development, suggesting a temporally specific negative regulatory mechanism controlled by PrePL in disease progression. Neither the truncated Pc, nor the full-length PrePL missing the Lon cleavage site complemented the ΔPrePL phenotype, suggesting that full-length PrePL and Pc both function in fungal development. PrePL targeted to the mitochondria undergoes hydrolysis by Lon to produce Pc, which accumulates in the fungal apoplast. Importantly, recombinant Pc induced plant defence responses and cell death after being infiltrated into selected plant leaves, indicating that it functions as an avirulence factor. This work thus reveals a novel pathogenic factor in the fungal Lon-mediated pathway. Additionally, our results provide new insight into the functions of a full-length protein and its cleaved isoform in fungal pathogenesis.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

In the Gram-positive pathogen Staphylococcus aureus, the membrane-bound ATP-dependent metalloprotease FtsH plays a critical role in resistance to various stressors. However, the molecular mechanism of the FtsH functions is not known. Here, we identified core FtsH target proteins in S. aureus. In the strains Newman and USA300, the abundance of 33 proteins were altered in both strains, of which 11 were identified as core FtsH substrate protein candidates. In the strain Newman and some other S. aureus strains, the sensor histidine kinase SaeS has an L18P (T53C in saeS) substitution, which transformed the protein into an FtsH substrate. Due to the increase of SaeS L18P in the ftsH mutant, Eap, a sae-regulon protein, was also increased in abundance, causing the Newman-specific cell-aggregation phenotype. Regardless of the strain background, however, the ftsH mutants showed lower virulence and survival in a murine infection model. Our study illustrates the elasticity of the bacterial regulatory network, which can be rewired by a single substitution mutation.

LpxC is a deacetylase that catalyzes the first committed step of lipid A biosynthesis in Escherichia coli. LpxC competes for a common precursor, R-3-hydroxymyristoyl-UDP-GlcNAc, with FabZ, whose dehydratase activity catalyzes the first committed step of phospholipid biosynthesis. To maintain the optimum flow of the common precursor to these two competing pathways, the LpxC level is controlled by FtsH/YciM-mediated proteolysis. It is not known whether this complex or another protein senses the status of lipid A synthesis to control LpxC proteolysis. The work carried out in this study began with a novel mutation, yejM1163, which causes hypersensitivity to large antibiotics such as vancomycin and erythromycin. Isolates resistant to these antibiotics carried suppressor mutations in the ftsH and yciM genes. Western blot analysis showed a dramatically reduced LpxC level in the yejM1163 background, while the presence of ftsH or yciM suppressor mutations restored LpxC levels to different degrees. Based on these observations, it is proposed that YejM is a sensor of lipid A synthesis and controls LpxC levels by modulating the activity of the FtsH/YciM complex. The truncation of the periplasmic domain in the YejM1163 protein causes unregulated proteolysis of LpxC, thus diverting a greater pool of R-3-hydroxymyristoyl-UDP-GlcNAc toward phospholipid synthesis. This imbalance in lipid synthesis perturbs the outer membrane permeability barrier, causing hypersensitivity toward vancomycin and erythromycin. yejM1163 suppressor mutations in ftsH and yciM lower the proteolytic activity toward LpxC, thus restoring lipid homeostasis and the outer membrane permeability barrier. IMPORTANCE Lipid homeostasis is critical for proper envelope functions. The level of LpxC, which catalyzes the first committed step of lipopolysaccharide (LPS) synthesis, is controlled by an essential protease complex comprised of FtsH and YciM. Work carried out here suggests YejM, an essential envelope protein, plays a central role in sensing the state of LPS synthesis and controls LpxC levels by regulating the activity of FtsH/YciM. All four essential proteins are attractive targets of therapeutic development.

LpxC is a deacetylase that catalyzes the first committed step of lipid A biosynthesis in Escherichia coli. LpxC competes for a common precursor, R-3-hydroxymyristoyl-UDP-GlcNAc, with FabZ, whose dehydratase activity catalyzes the first committed step of phospholipid biosynthesis. To maintain the optimum flow of the common precursor to these two competing pathways, the LpxC level is controlled by FtsH/YciM-mediated proteolysis. It is not known whether this complex or another protein senses the status of lipid A synthesis to control LpxC proteolysis. The work carried out in this study began with a novel mutation, yejM1163, which causes hypersensitivity to large antibiotics such as vancomycin and erythromycin. Isolates resistant to these antibiotics carried suppressor mutations in the ftsH and yciM genes. Western blot analysis showed a dramatically reduced LpxC level in the yejM1163 background, while the presence of ftsH or yciM suppressor mutations restored LpxC levels to different degrees. Based on these observations, it is proposed that YejM is a sensor of lipid A synthesis and controls LpxC levels by modulating the activity of the FtsH/YciM complex. The truncation of the periplasmic domain in the YejM1163 protein causes unregulated proteolysis of LpxC, thus diverting a greater pool of R-3-hydroxymyristoyl-UDP-GlcNAc toward phospholipid synthesis. This imbalance in lipid synthesis perturbs the outer membrane permeability barrier, causing hypersensitivity toward vancomycin and erythromycin. yejM1163 suppressor mutations in ftsH and yciM lower the proteolytic activity toward LpxC, thus restoring lipid homeostasis and the outer membrane permeability barrier. IMPORTANCE Lipid homeostasis is critical for proper envelope functions. The level of LpxC, which catalyzes the first committed step of lipopolysaccharide (LPS) synthesis, is controlled by an essential protease complex comprised of FtsH and YciM. Work carried out here suggests YejM, an essential envelope protein, plays a central role in sensing the state of LPS synthesis and controls LpxC levels by regulating the activity of FtsH/YciM. All four essential proteins are attractive targets of therapeutic development.

Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type 28 (SCA28) or recessive spastic ataxia with epilepsy (SPAX5). Functional studies in patient fibroblasts demonstrate that the p.G337E AFG3L2 mutation strongly destabilizes the long isoforms of OPA1 via OMA hyper-activation and leads to mitochondrial fragmentation, thus explaining the family phenotype. This study widens the clinical spectrum of neurodegenerative diseases caused by AFG3L2 mutations, which shall be considered as genetic cause of ADOA.

RNA modifications affect the stability and function of RNA species, regulating important downstream processes. Modification levels are often dynamic, varying between tissues and individuals, although it is not always clear what modulates this or what impact it has on biological systems. Here, we quantify variation in m1A/G RNA modification levels at functionally important positions in the human mitochondrial genome across 11,552 samples from 39 tissue/cell types and find that modification levels are associated with mitochondrial transcript processing. We identify links between mitochondrial RNA modification levels and genetic variants in the nuclear genome, including a missense mutation in LONP1, and find that genetic variants within MRPP3 and TRMT61B are associated with RNA modification levels across a large number of tissues. Genetic variants linked to RNA modification levels are associated with multiple disease/disease-related phenotypes, including blood pressure, breast cancer and psoriasis, suggesting a role for mitochondrial RNA modification in complex disease., Ali et al. analyze publicly available RNA-seq data from different tissues to quantify variation in m1A/G methylation levels in mitochondrial RNAs. They show a link between mitochondrial m1A/G modification levels and nuclear genetic variants, many of which are associated with disease.

Background Metastasis causes the vast majority of colorectal carcinoma (CRC)-related deaths. However, little is known about the specific traits and underlying mechanisms of metastasis-initiating cells in primary CRC. And whether or not circular RNAs (circRNAs) take part in this particular event remain not adequately stated yet. Methods A screening method based on Transwell assay was first applied to build CRC subgroups with different metastatic potential. High throughput RNA sequencing was used to find out novel metastatic drivers in CRC metastasis-initiating step. A series of in vitro and in vivo assays were further applied to elucidate the functions and underlying molecular mechanisms of circRNAs in CRC metastasis. Results A circRNA consisting of exon 8–11 of LONP2, termed as circLONP2, was upregulated in metastasis-initiating CRC subgroups. Aberrant higher expression of circLONP2 was observed in primary CRC tissues with established metastasis, and along the invasive margin in metastatic site. High expression of circLONP2 predicted unfavorable overall survival. Functional studies revealed that circLONP2 could enhance the invasiveness of CRC cells in vitro, and targeting circLONP2 through anti-sense oligonucleotide (ASO) dramatically reduced the penetrance of metastasis to foreign organs in vivo. Mechanically, circLONP2 directly interacted with and promoted the processing of primary microRNA-17 (pri-miR-17), through recruiting DiGeorge syndrome critical region gene 8 (DGCR8) and Drosha complex in DDX1-dependent manner. Meanwhile, upregulated mature miR-17-5p could be assembled into exosomes and internalized by neighboring cells to enhance their aggressiveness. Conclusions Our data indicate that circLONP2 acts as key metastasis-initiating molecule during CRC progression through modulating the intracellular maturation and intercellular transfer of miR-17, resulting in dissemination of metastasis-initiating ability in primary site and acceleration of metastasis formation in foreign organs. circLONP2 could serve as an effective prognostic predictor and/or novel anti-metastasis therapeutic target in CRC treatment.

Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.

Mitochondrial function depends crucially on the maintenance of multiple mitochondrial DNA (mtDNA) copies. Surprisingly, the cellular mechanisms regulating mtDNA copy number remain poorly understood. Through a systematic high-throughput approach in Saccharomyces cerevisiae, we determined mtDNA-to-nuclear DNA ratios in 5148 strains lacking nonessential genes. The screen revealed MRX6, a largely uncharacterized gene, whose deletion resulted in a marked increase in mtDNA levels, while maintaining wild type-like mitochondrial structure and cell size. Quantitative superresolution imaging revealed that deletion of MRX6 alters both the size and the spatial distribution of mtDNA nucleoids. We demonstrate that Mrx6 partially colocalizes with mtDNA within mitochondria and interacts with the conserved Lon protease Pim1 in a complex that also includes Mam33 and the Mrx6-related protein Pet20. Acute depletion of Pim1 phenocopied the high mtDNA levels observed in Δmrx6 cells. No further increase in mtDNA copy number was observed upon depletion of Pim1 in Δmrx6 cells, revealing an epistatic relationship between Pim1 and Mrx6. Human and bacterial Lon proteases regulate DNA replication by degrading replication initiation factors, suggesting a model in which Pim1 acts similarly with the Mrx6 complex, providing a scaffold linking it to mtDNA.

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C > T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings’ fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings’ fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings’ fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Cereblon (CRBN) has been reported as a negative regulator of adenosine monophosphate-activated protein kinase (AMPK). Aerobic exercise training has been shown to increase AMPK, which resulted in glucose regulation in skeletal muscle. However, the expression level of CRBN and its association with the physiological modulation of glucose are still unclear. Male Sprague-Dawley rats (5-week-old, n = 18) were assigned to control, streptozotocin (STZ, 65 mg/kg)-induced diabetic group, and STZ + exercise (STZ + EXE) group with six rats in each group. Rats in the STZ + EXE group exercised by treadmill running (20 m/min, 60 min, 4 times/week) for 8 weeks. Compared with the STZ group, blood glucose was significantly decreased in the STZ + EXE group. The skeletal muscle of rats in the STZ + EXE group showed a significant decrease in CRBN levels and an increase in AMPK, protein kinase B, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, fibronectin type III domain-containing protein 5, glucose transporter type 4, superoxide dismutase 1, and uncoupling protein 3 levels. These results suggest that CRBN is a potential regulator of glucose homeostasis in the skeletal muscle. Moreover, our results suggest that aerobic exercise training may provide an important physiological treatment for type 1 diabetes by decreasing CRBN and increasing AMPK signaling in skeletal muscle.

Thalidomide was originally used as a sedative and found to be a teratogen, but now thalidomide and its derivatives are widely used to treat haematologic malignancies. Accumulated evidence suggests that thalidomide suppresses nerve cell death in neurologic model mice. However, detailed molecular mechanisms are unknown. Here we examined the molecular mechanism of thalidomide’s neuroprotective effects, focusing on its target protein, cereblon (CRBN), and its binding protein, AMP-activated protein kinase (AMPK), which plays an important role in maintaining intracellular energy homeostasis in the brain. We used a cerebral ischemia rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). Thalidomide treatment significantly decreased the infarct volume and neurological deficits of MCAO/R rats. AMPK was the key signalling protein in this mechanism. Furthermore, we considered that the AMPK–CRBN interaction was altered when neuroprotective action by thalidomide occurred in cells under ischemic conditions. Binding was strong between AMPK and CRBN in normal SH-SY5Y cells, but was weakened by the addition of H2O2. However, when thalidomide was administered at the same time as H2O2, the binding of AMPK and CRBN was partly restored. These results suggest that thalidomide inhibits the activity of AMPK via CRBN under oxidative stress and suppresses nerve cell death.

The turnover of mitochondrial outer-membrane proteins is known to be mediated by the cytoplasmic ubiquitin–proteasome pathway. Wu et al. report the unexpected finding that two outer-membrane proteins Tom22 and Om45 are inwardly translocated into mitochondria and degraded by the inner-membrane protease Yme1., Mitochondria are double-membraned organelles playing essential metabolic and signaling functions. The mitochondrial proteome is under surveillance by two proteolysis systems: the ubiquitin–proteasome system degrades mitochondrial outer-membrane (MOM) proteins, and the AAA proteases maintain the proteostasis of intramitochondrial compartments. We previously identified a Doa1–Cdc48-Ufd1-Npl4 complex that retrogradely translocates ubiquitinated MOM proteins to the cytoplasm for degradation. In this study, we report the unexpected identification of MOM proteins whose degradation requires the Yme1-Mgr1-Mgr3 i-AAA protease complex in mitochondrial inner membrane. Through immunoprecipitation and in vivo site-specific photo–cross-linking experiments, we show that both Yme1 adapters Mgr1 and Mgr3 recognize the intermembrane space (IMS) domains of the MOM substrates and facilitate their recruitment to Yme1 for proteolysis. We also provide evidence that the cytoplasmic domain of substrate can be dislocated into IMS by the ATPase activity of Yme1. Our findings indicate a proteolysis pathway monitoring MOM proteins from the IMS side and suggest that the MOM proteome is surveilled by mitochondrial and cytoplasmic quality control machineries in parallel.

Background Mutations in AFG3L2 , a gene encoding a subunit of the mitochondrion m-AAA protease, cause spinocerebellar ataxia type 28 and recessive spastic ataxia type 5. Neuroimaging shows cerebellar atrophy. Methods Retrospective review of the patient charts including their clinical evaluation and molecular genetic, neurodiagnostic, and neuroradiological investigations. Results We describe five members of a large consanguineous family with a severe mitochondrial disease phenotype in the form of regression of the developmental milestones in the first year of life, refractory epilepsy, progressive microcephaly, increased blood lactate, basal ganglia involvement, and premature death. Exome sequencing showed homozygous mutation of the AFG3L2 gene in all individuals: c.1714G>A (p.Ala572Thr). Conclusions Our findings add to the phenotypic, neuroradiological, genetic, and biochemical spectrum of AFG3L2 mutations.

Mitochondria have many functions, including ATP generation. The electron transport chain (ETC) and the coupled ATP synthase generate ATP by consuming oxygen. Reactive oxygen species (ROS) are also produced by ETC, and ROS damage deoxyribonucleic acids, membrane lipids and proteins. Recent analysis indicate that mitochondrial unfolded protein response (UPRmt), which enhances expression of mitochondrial chaperones and proteases to remove damaged proteins, is activated when damaged proteins accumulate in the mitochondria. In Caenorhabditis elegans, HAF-1, a putative ortholog of human ABCB10, plays an essential role in signal transduction from mitochondria to nuclei to enhance UPRmt. Therefore, it is possible that ABCB10 has a role similar to that of HAF-1. However, it has not been reported whether ABCB10 is a factor in the signal transduction pathway to enhance UPRmt. In this study, ABCB10 was depleted in HepG2 cells using small interfering RNA (siRNA), and the effect was examined. ABCB10 depletion upregulated ROS and the expression of ROS-detoxifying enzymes (SOD2, GSTA1, and GSTA2), and SESN3, a protein induced by ROS to protect the cell from oxidative stress. In addition, ABCB10 depletion significantly decreased expression of UPRmt-related mitochondrial chaperones (HSPD1 and DNAJA3), and a mitochondrial protease (LONP1). However, the putative activity of ABCB10 to export peptides from mitochondria was not lost by ABCB10 depletion. Altogether, these data suggest that ABCB10 is involved in UPRmt signaling pathway similar to that of HAF-1, although ABCB10 probably does not participate in peptide export from mitochondria.

The mitochondrial unfolded protein response (UPRmt) is a transcriptional program aimed at restoring proteostasis in mitochondria. Upregulation of mitochondrial matrix proteases and heat shock proteins was initially described. Soon thereafter, a distinct UPRmt induced by misfolded proteins in the mitochondrial intermembrane space (IMS) and mediated by the estrogen receptor alpha (ERα), was found to upregulate the proteasome and the IMS protease OMI. However, the IMS-UPRmt was never studied in a neurodegenerative disease in vivo. Thus, we investigated the IMS-UPRmt in the G93A-SOD1 mouse model of familial ALS, since mutant SOD1 is known to accumulate in the IMS of neural tissue and cause mitochondrial dysfunction. As the ERα is most active in females, we postulated that a differential involvement of the IMS-UPRmt could be linked to the longer lifespan of females in the G93A-SOD1 mouse. We found a significant sex difference in the IMS-UPRmt, because the spinal cords of female, but not male, G93A-SOD1 mice showed elevation of OMI and proteasome activity. Then, using a mouse in which G93A-SOD1 was selectively targeted to the IMS, we demonstrated that the IMS-UPRmt could be specifically initiated by mutant SOD1 localized in the IMS. Furthermore, we showed that, in the absence of ERα, G93A-SOD1 failed to activate OMI and the proteasome, confirming the ERα dependence of the response. Taken together, these results demonstrate the IMS-UPRmt activation in SOD1 familial ALS, and suggest that sex differences in the disease phenotype could be linked to differential activation of the ERα axis of the IMS-UPRmt.

Neuropathies caused by mitochondrial dysfunction are the most common and serious impediment of high glucose (HG)-induced toxicity. We have previously reported mitoprotective potency of Sirtuin1 (Sirt1) in diabetic neuropathy (DN) via targeting mitochondrial dysfunction but its nuclear control over mitochondrial bioenergetics remains unknown. Here, we studied the effect of SRT1720; a small molecule activator of Sirt1 in attenuating the HG mediated mitochondrial dysfunction in differentiated rat pheochromocytoma (PC12) cells and aiming to determine (1) whether SRT1720 can improve mitochondrial function in HG exposed PC12 cells (2) if yes then this effect is dependent or independent of mitochondrial Lon protease (LONP1) (3) and whether silencing of LONP1 affects the mitochondrial function or not. HG (30 mM) exposed PC12 cells demonstrated reduced mitochondrial complex activities and oxygen consumption rate (OCR), decreased the expressions of Sirt1, peroxisome proliferator-activated receptor coactivator-1α (PGC1α), nuclear respiratory factor-2 (NRF2), LONP1 and ATP synthase c. SRT1720 treatment (4 μM) significantly reversed these effects in hyperglycemia insulted PC12 cells but silencing the expression of LONP1 impeded this effect of SRT1720 on mitochondrial complex activities, OCR and mitochondrial membrane potential. Based on these findings, we inferred that SRT1720 might improve mitochondrial function in HG induced mitochondrial dysfunction in PC12 cells via stimulation of Sirt1-LONP1 axis.

Diabetes is a metabolic disorder associated with mitochondrial (mt) dysfunction and oxidative stress. The molecular mechanisms involved in diabetes-associated neurological complications remain elusive. This study aims to investigate the protective effect of metformin (MF) on regulatory networks and integrated stress responses in brain tissue of Streptozotocin (STZ)-induced diabetic mice. STZ-induced diabetic mice were treated with MF (20 mg/kg BW), and whole brain tissue was harvested for further analysis. Protein carbonylation was measured as a marker of neuronal oxidative stress. Protein expression of mt chaperones, maintenance proteins, and regulators of the unfolded protein response (UPR) were measured by Western blot. Transcript levels of antioxidant enzyme GSTA4; mt biogenesis markers, ER stress regulators, and miR-132 and miR-148a were analysed using qPCR. The results showed that MF efficiently reduced protein carbonylation and oxidation. Mt function was improved by MF-treatment through upregulation of chaperone proteins (HSP60, HSP70 and LonP1). MF elicits the UPR to attenuate ER stress through a miR-132 repression mechanism. Additionally, MF was found to elevate deacetylases- Sirt1, Sirt3; and mt biogenesis marker PGC-1α through miR-148a repression. This is the first study to demonstrate the epigenetic regulation of mt maintenance by MF in diabetic C57BL/6 mouse whole brain tissue. We thus conclude that MF, beyond its anti-hyperglycaemic role, mediates neuroprotection through epigenomic and integrated stress responses in diabetic mice.

The haploinsufficiency of C9orf72 is implicated in the most common forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the full spectrum of C9orf72 functions remains to be established. Here, we report that C9orf72 is a mitochondrial inner-membrane-associated protein regulating cellular energy homeostasis via its critical role in the control of oxidative phosphorylation (OXPHOS). The translocation of C9orf72 from the cytosol to the inter-membrane space is mediated by the redox-sensitive AIFM1/CHCHD4 pathway. In mitochondria, C9orf72 specifically stabilizes translocase of inner mitochondrial membrane domain containing 1 (TIMMDC1), a crucial factor for the assembly of OXPHOS complex I. C9orf72 directly recruits the prohibitin complex to inhibit the m-AAA protease-dependent degradation of TIMMDC1. The mitochondrial complex I function is impaired in C9orf72-linked ALS/FTD patient-derived neurons. These results reveal a previously unknown function of C9orf72 in mitochondria and suggest that defective energy metabolism may underlie the pathogenesis of relevant diseases.

Singlet oxygen ((1)O(2)) is a potent oxidizing agent, principally generated by photosystem II (PSII) as a byproduct of photosynthesis. Hence, (1)O(2) damages PSII, especially the PSII reaction center (RC) proteins, promoting a process called PSII repair cycle. The hetero-hexameric FtsH protease, located in the thylakoid membrane, is essential in degrading these damaged PSII RC proteins, which defines the first step of the PSII repair. The loss of the central subunit of the FtsH protease, FtsH2 (VAR2), weakens the PSII repair, thereby impairing PSII proteostasis. A recent study demonstrated that the impaired proteostasis (or accumulation of damaged proteins) in the chloroplasts of the var2 mutant induces an unfolded/misfolded protein response (UPR)-like response, more appropriately referred to as a damaged protein response (DPR), as evident in the accumulation of proteins related to the protein quality control (PQC). Comparison of data from chloroplast proteomics data with RNA sequencing in the context of the UPR-like response suggests a plausible activation of retrograde signaling in the var2 mutant. Either through the enhanced level of (1)O(2) or by impairing the substrate-unfolding activity of FtsH2, the reinforced defect appears to induce stress-related genes via the stress hormone salicylic acid (SA). This finding suggests that impaired chloroplast proteostasis (specifically for PSII proteins) may activate the chloroplast-established isochorismate pathway to produce SA. If this assumption is correct, then SA serves as a retrograde signaling molecule. In this review, we will discuss the impact of chloroplast proteostasis on chloroplasts-to-nucleus communication.

LONP1 is an ATP-dependent protease and chaperone that plays multiple vital roles in mitochondria. LONP1 is essential for mitochondrial homeostasis due to its role in maintenance of the mitochondrial genome and its central role in regulating mitochondrial processes such as oxidative phosphorylation, mitophagy, and heme biosynthesis. Bi-allelic LONP1 mutations have been reported to cause a constellation of clinical presentations. We report a patient heterozygous for a de novo mutation in LONP1: c.901C>T,p.R301W presenting as a neonate with seizures, encephalopathy, pachygyria and microcephaly. Assays of respiratory chain activity in muscle showed complex II-III function at 8% of control. Functional studies in patient fibroblasts showed a signature of dysfunction that included significant decreases in known proteolytic targets of LONP1 (TFAM, PINK1, phospho-PDH E1α) as well as loss of mitochondrial ribosome subunits MRPL44 and MRPL11 with concomitant decreased activity and level of protein subunits of oxidative phosphorylation complexes I and IV. These results indicate excessive LONP1 proteolytic activity and a loss of LONP1 chaperone activity. Further, we demonstrate that the LONP1 N-terminal domain is involved in hexamer stability of LONP1 and that the ability to make conformational changes is necessary for LONP1 to regulate proper functioning of both its proteolytic and chaperone activities.

Mitochondrial LonP1 is an essential stress response protease that mediates mitochondrial proteostasis, metabolism and bioenergetics. Homozygous and compound heterozygous variants in the LONP1 gene encoding the LonP1 protease have recently been shown to cause a diverse spectrum of human pathologies, ranging from classical mitochondrial disease phenotypes, profound neurologic impairment and multi-organ dysfunctions, some of which are uncommon to mitochondrial disorders. In this review, we focus primarily on human LonP1 and discuss findings, which demonstrate its multidimensional roles in maintaining mitochondrial proteostasis and adapting cells to metabolic flux and stress during normal physiology and disease processes. We also discuss emerging roles of LonP1 in responding to developmental, oncogenic and cardiac stress.

Summary The proteolytic turnover of mitochondrial proteins is poorly understood. Here, we used a combination of dynamic isotope labeling and mass spectrometry to gain a global overview of mitochondrial protein turnover in yeast cells. Intriguingly, we found an exceptionally high turnover of the NADH dehydrogenase, Nde1. This homolog of the mammalian apoptosis inducing factor, AIF, forms two distinct topomers in mitochondria, one residing in the intermembrane space while the other spans the outer membrane and is exposed to the cytosol. The surface-exposed topomer triggers cell death in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1; however, it is strongly enriched in respiratory-deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised cells from growing populations.

Objective The aim of this study was to investigate the role of LONP1 in the progression of pancreatic cancer. Methods Lentivirus was used to silence LONP1 in PANC-1 cells. Colony formation assay, cell counting kit (CCK8) assay, cell scratch-wound assay, and transwell assay were used to assess the effects of our strategy on inhibiting cancer growth, migration, and invasion. Protein expression was detected by Western blot analysis. Results The expression of LONP1 in pancreatic carcinoma tissues was higher than that in adjacent normal pancreatic tissues. Downregulation of LONP1 suppressed the proliferation, migration, and invasion of PANC-1 cells. Knockdown of LONP1 in PANC-1 cells inhibited epithelial-mesenchymal transition and matrix metalloprotein (MMP) 2/9 by downregulation of vimentin, snail, slug, MMP2, and MMP9 and upregulation of claudin-1. The c-Jun N-terminal kinase pathway was inactivated in LONP1 knockdown PANC-1 cells. Activation of the c-Jun N-terminal kinase pathway by anisomycin treatment significantly reversed the changes in epithelial-mesenchymal transition markers and MMP2/9 induced by ablation of LONP1 in PANC-1 cells. Conclusions LONP1 plays a vital role in the proliferation and metastasis of pancreatic cancer, which provides a potential therapeutic target for the treatment of pancreatic cancer.

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases., Author summary TDP-43 proteinopathy is a group of fatal neurological diseases. Here, we report a systematic examination of the role of mitochondrial damage in TDP-43 proteinopathy using patient brain tissues, as well as cellular and animal models. Our data show that TDP-43 induces severe mitochondrial damage, accompanied by activation of UPRmt in both cellular and animal models of TDP-43 proteinopathy. LonP1, one of the key mitochondrial proteases in UPRmt, protects against TDP-43 induced cytotoxicity and neurodegeneration. Our study uncovers LonP1 as a modifier gene for TDP-43 proteinopathy and suggests protecting against or reversing mitochondrial damage as a potential therapeutic approach to these neurodegenerative disorders.

Mitochondrial AAA+ quality control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying molecular mechanisms that define the diverse activities of these enzymes remain poorly defined. The mitochondrial AAA+ protease AFG3L2 is of particular interest, as genetic mutations localized throughout AFG3L2 are linked to diverse neurodegenerative disorders. However, a lack of structural data has limited our understanding of how mutations impact enzymatic activity. Here, we used cryo-EM to determine a substrate-bound structure of the catalytic core of human AFG3L2. This structure identifies multiple specialized structural features within AFG3L2 that integrate with conserved structural motifs required for hand-over-hand ATP-dependent substrate translocation to engage, unfold and degrade targeted proteins. Mapping disease-relevant AFG3L2 mutations onto our structure demonstrates that many of these mutations localize to these unique structural features of AFG3L2 and distinctly influence its activity and stability. Our results provide a molecular basis for neurological phenotypes associated with different AFG3L2 mutations, and establish a structural framework to understand how different members of the AAA+ superfamily achieve specialized, diverse biological functions.

Research on healthy aging shows that lifespan reductions are often caused by mitochondrial dysfunction. Thus, it is very interesting that the deletion of mitochondrial matrix peptidase LonP1 was observed to abolish embryogenesis, while deletion of the mitochondrial matrix peptidase Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (ClpP) prolonged survival. To unveil the targets of each enzyme, we documented the global proteome of LonP1+/&minus, mouse embryonal fibroblasts (MEF), for comparison with ClpP&minus, /&minus, depletion. Proteomic profiles of LonP1+/&minus, MEF generated by label-free mass spectrometry were further processed with the STRING (Search tool for the retrieval of interacting genes) webserver Heidelberg for protein interactions. ClpP was previously reported to degrade Eral1 as a chaperone involved in mitoribosome assembly, so ClpP deficiency triggers the accumulation of mitoribosomal subunits and inefficient translation. LonP1+/&minus, MEF also showed Eral1 accumulation, but no systematic effect on mitoribosomal subunits. In contrast to ClpP&minus, profiles, several components of the respiratory complex-I membrane arm, of the glutathione pathway and of lysosomes were accumulated, whereas the upregulation of numerous innate immune defense components was similar. Overall, LonP1, as opposed to ClpP, appears to have no effect on translational machinery, instead it shows enhanced respiratory dysfunction, this agrees with reports on the human CODAS syndrome (syndrome with cerebral, ocular, dental, auricular, and skeletal anomalies) caused by LonP1 mutations.

BackgroundSpinocerebellar ataxia type 28 (SCA28) is a dominantly inherited neurodegenerative disease caused by pathogenic variants in AFG3L2. The AFG3L2 protein is a subunit of mitochondrial m-AAA complexes involved in protein quality control. Objective of this study was to determine the molecular mechanisms of SCA28, which has eluded characterisation to date.MethodsWe derived SCA28 patient fibroblasts carrying different pathogenic variants in the AFG3L2 proteolytic domain (missense: the newly identified p.F664S and p.M666T, p.G671R, p.Y689H and a truncating frameshift p.L556fs) and analysed multiple aspects of mitochondrial physiology. As reference of residual m-AAA activity, we included SPAX5 patient fibroblasts with homozygous p.Y616C pathogenic variant, AFG3L2+/− HEK293 T cells by CRISPR/Cas9-genome editing and Afg3l2−/− murine fibroblasts.ResultsWe found that SCA28 cells carrying missense changes have normal levels of assembled m-AAA complexes, while the cells with a truncating pathogenic variant had only half of this amount. We disclosed inefficient mitochondrial fusion in SCA28 cells caused by increased OPA1 processing operated by hyperactivated OMA1. Notably, we found altered mitochondrial proteostasis to be the trigger of OMA1 activation in SCA28 cells, with pharmacological attenuation of mitochondrial protein synthesis resulting in stabilised levels of OMA1 and OPA1 long forms, which rescued mitochondrial fusion efficiency. Secondary to altered mitochondrial morphology, mitochondrial calcium uptake resulted decreased in SCA28 cells.ConclusionOur data identify the earliest events in SCA28 pathogenesis and open new perspectives for therapy. By identifying similar mitochondrial phenotypes between SCA28 cells and AFG3L2+/− cells, our results support haploinsufficiency as the mechanism for the studied pathogenic variants.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significant. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was also altered, in line with greatly reduced expression of Opa1 fusogenic protein L-isoforms. The mitochondrial alterations observed in MEFs were also detected in cerebella of 18-month-old heterozygous mutants, suggesting they may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Proteases play essential roles in cellular proteostasis. Mechanisms through which proteases recognize their substrates are often hard to predict and therefore require experimentation. In vivo trapping allows systematic identification of potential substrates of proteases, their adaptors, and chaperones. This combines in vivo genetic modifications of proteolytic systems, stabilized protease–substrate interactions, affinity enrichments of trapped substrates, and mass spectrometry (MS)-based identification. In vitro approaches, in which immobilized protease components are incubated with isolated cellular proteome, complement this in vivo approach. Both approaches can provide information about substrate recognition signals, degrons, and conditional effects. This review summarizes published trapping studies and their biological outcomes, and provides recommendations for substrate trapping of the processive AAA+ Clp, Lon, and FtsH chaperone proteolytic systems.

Lon protease 1 (LONP1) is a highly conserved serine peptidase that plays an important role in the protein quality control system in mammalian mitochondria. LONP1 catalyzes the degradation of oxidized, dysfunctional, and misfolded matrix proteins inside mitochondria and regulates mitochondrial gene expression and genome integrity. Therefore, LONP1 is up-regulated and suppresses cell death in response to oxidative stress, heat shock, and nutrient starvation. On the other hand, translocation of high mobility group box 1 (HMGB1) and active caspase-3 into mitochondria is involved in apoptosis of parvalbumin (PV) cells (one of the GABAergic interneurons) and necrosis of CA1 neurons in the rat hippocampus, respectively, following status epilepticus (SE). In the present study, we investigated whether LONP1 may improve neuronal viability to prevent or ameliorate translocation of active caspase-3 and HMGB1 in mitochondria within PV and CA1 neurons. Following SE, LONP1 expression was up-regulated in mitochondria of PV and CA1 neurons. LONP1 knockdown deteriorated SE-induced neuronal death with mitochondrial accumulation of active caspase-3 and HMGB1 in PV cells and CA1 neurons, respectively. LONP1 knockdown did not affect the aberrant mitochondrial machinery induced by SE. Therefore, our findings suggest, for the first time, that LONP1 may contribute to the alleviation of mitochondrial overloads of active caspase-3 and HMGB1, and the maintenance of neuronal viability against SE.

Isoform 1 of uracil-DNA glycosylase (UNG1) is the major protein for initiating base-excision repair in mitochondria and is in close proximity to the respiratory chain that generates reactive oxygen species (ROS). Effects of ROS on the stability of UNG1 have not been well characterized. In the present study, we found that overexpression of UNG1 enhanced cells' resistance to oxidative stress and protected mitochondrial DNA (mtDNA) from oxidation. Proteomics analysis showed that UNG1 bound to eight proteins in the mitochondria, including PAPSS2, CD70 antigen, and AGR2 under normal growth conditions, whereas UNG1 mainly bound to Peroxiredoxin 3 (PRDX3) via a disulfide linkage under oxidative stress. We further demonstrated that the UNG1-PRDX3 interaction protected UNG1 from ROS-mediated degradation and prevented mtDNA oxidation. Moreover, our results show that ROS-mediated UNG1 degradation was Lon protease 1 (LonP1)-dependent and mitochondrial UNG1 degradation was aggravated by knockdown of PRDX3 expression. Taken together, these results reveal a novel function of UNG1 in the recruitment of PRDX3 to mtDNA under oxidative stress, enabling protection of UNG1 and UNG1-bound DNA from ROS damage and enhancing cell resistance to oxidative stress.

In eubacteria, the tmRNA system frees ribosomes that stall during protein synthesis and adds an ssrA tag to the incompletely translated polypeptide to target it for degradation. The AAA+ ClpXP protease degrades most ssrA-tagged proteins in the Escherichia coli cytoplasm and was recently shown to degrade an ssrA-tagged protein in the inner membrane. However, we find that tmRNA-mediated tagging of E. coli ProW1–182, a different inner-membrane protein, results in degradation by the membrane-tethered AAA+ FtsH protease. ClpXP played no role in the degradation of ProW1–182 in vivo. These studies suggest that a complex distribution of proteolytic labor maintains protein quality control in the inner membrane.