m6A-methylated Lonp1 drives mitochondrial proteostasis stress to induce testicular pyroptosis upon environmental cadmium exposure.

Cadmium (Cd) is a widely distributed typical environmental pollutant and one of the most toxic heavy metals. It is well-known that environmental Cd causes testicular damage by inducing classic types of cell death such as cell apoptosis and necrosis. However, as a new type of cell death, the role and mechanism of pyroptosis in Cd-induced testicular injury remain unclear. In the current study, we used environmental Cd to generate a murine model with testicular injury and AIM2-dependent pyroptosis. Based on the model, we found that increased cytoplasmic mitochondrial DNA (mtDNA), activated mitochondrial proteostasis stress occurred in Cd-exposed testes. We used ethidium bromide to generate mtDNA-deficient testicular germ cells and further confirmed that increased cytoplasmic mtDNA promoted AIM2-dependent pyroptosis in Cd-exposed cells. Uracil-DNA glycosylase UNG1 overexpression indicated that environmental Cd blocked UNG-dependent repairment of damaged mtDNA to drive the process in which mtDNA releases to cytoplasm in the cells. Interestingly, we found that environmental Cd activated mitochondrial proteostasis stress by up-regulating protein expression of LONP1 in testes. Testicular specific LONP1-knockdown significantly reversed Cd-induced UNG1 protein degradation and AIM2-dependent pyroptosis in mouse testes. In addition, environmental Cd significantly enhanced the m6A modification of Lonp1 mRNA and its stability in testicular germ cells. Knockdown of IGF2BP1, a reader of m6A modification, reversed Cd-induced upregulation of LONP1 protein expression and pyroptosis activation in testicular germ cells. Collectively, environmental Cd induces m6A modification of Lonp1 mRNA to activate mitochondrial proteostasis stress, increase cytoplasmic mtDNA content, and trigger AIM2-dependent pyroptosis in mouse testes. These findings suggest that mitochondrial proteostasis stress is a potential target for the prevention of testicular injury.

Lon1 Inactivation Downregulates Autophagic Flux and Brassinosteroid Biogenesis, Modulating Mitochondrial Proportion and Seed Development in Arabidopsis.

Mitochondrial protein homeostasis is crucially regulated by protein degradation processes involving both mitochondrial proteases and cytosolic autophagy. However, it remains unclear how plant cells regulate autophagy in the scenario of lacking a major mitochondrial Lon1 protease. In this study, we observed a notable downregulation of core autophagy proteins in Arabidopsis Lon1 knockout mutant lon1-1 and lon1-2, supporting the alterations in the relative proportions of mitochondrial and vacuolar proteins over total proteins in the plant cells. To delve deeper into understanding the roles of the mitochondrial protease Lon1 and autophagy in maintaining mitochondrial protein homeostasis and plant development, we generated the lon1-2atg5-1 double mutant by incorporating the loss-of-function mutation of the autophagy core protein ATG5, known as atg5-1. The double mutant exhibited a blend of phenotypes, characterized by short plants and early senescence, mirroring those observed in the individual single mutants. Accordingly, distinct transcriptome alterations were evident in each of the single mutants, while the double mutant displayed a unique amalgamation of transcriptional responses. Heightened severity, particularly evident in reduced seed numbers and abnormal embryo development, was observed in the double mutant. Notably, aberrations in protein storage vacuoles (PSVs) and oil bodies were evident in the single and double mutants. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of genes concurrently downregulated in lon1-2, atg5-1, and lon1-2atg5-1 unveiled a significant suppression of genes associated with brassinosteroid (BR) biosynthesis and homeostasis. This downregulation likely contributes to the observed abnormalities in seed and embryo development in the mutants.

[Protein degradation in bacteria: focus on the ClpP protease].

Proteins in the cells are born (synthesized), work, and die (decomposed). In the life of a protein, its birth is obviously important, but how it dies is equally important in living organisms. Proteases secreted into the outside of cells are used to decompose the external proteins and the degradation products are taken as the nutrients. On the other hand, there are also proteases that decompose unnecessary or harmful proteins which are generated in the cells. In eukaryotes, a large enzyme complex called the proteasome is primarily responsible for degradation of such proteins. Bacteria, which are prokaryotes, have a similar system as the proteasome. We would like to explain the bacterial degradation system of proteins or the death of proteins, which is performed by ATP-dependent protease Clp, with a particular focus on the ClpXP complex, and with an aspect as a target for antibiotics against bacteria.

The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C.

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

LONP1 ameliorates liver injury and improves gluconeogenesis dysfunction in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown. METHODS: In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF. RESULTS: We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders. CONCLUSION: We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.

AFG3L2 and ACO2-Linked Dominant Optic Atrophy: Genotype-Phenotype Characterization Compared to OPA1 Patients.

PURPOSE: Heterozygous mutations in the AFG3L2 gene (encoding a mitochondrial protease indirectly reflecting on OPA1 cleavage) and ACO2 gene (encoding the mitochondrial enzyme aconitase) are associated with isolated forms of Dominant Optic Atrophy (DOA). We aimed at describing their neuro-ophthalmological phenotype as compared with classic OPA1-related DOA. DESIGN: Cross-sectional study. METHODS: The following neuro-ophthalmological parameters were collected: logMAR visual acuity (VA), color vision, mean deviation and foveal threshold at visual fields, average and sectorial retinal nerve fiber layer (RNFL), and ganglion cell layer (GCL) thickness on optical coherence tomography. ACO2 and AFG3L2 patients were compared with an age- and sex-matched group of OPA1 patients with a 1:2 ratio. All eyes were analyzed using a clustered Wilcoxon rank sum test with the Rosner-Glynn-Lee method. RESULTS: A total of 44 eyes from 23 ACO2 patients and 26 eyes from 13 AFG3L2 patients were compared with 143 eyes from 72 OPA1 patients. All cases presented with bilateral temporal-predominant optic atrophy with various degree of visual impairment. Comparison between AFG3L2 and OPA1 failed to reveal any significant difference. ACO2 patients compared to both AFG3L2 and OPA1 presented overall higher values of nasal RNFL thickness (P = .029, P = .023), average thickness (P = .012, P = .0007), and sectorial GCL thickness. These results were confirmed also comparing separately affected and subclinical patients. CONCLUSIONS: Clinically, DOA remains a fairly homogeneous entity despite the growing genetic heterogeneity. ACO2 seems to be associated with an overall better preservation of retinal ganglion cells, probably depending on the different pathogenic mechanism involving mtDNA maintenance, as opposed to AFG3L2, which is involved in OPA1 processing and is virtually indistinguishable from classic OPA1-DOA.

Mitochondrial translocation of TFEB regulates complex I and inflammation.

TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis.

Organelle transporters define metabolic compartmentalization, and how this metabolite transport process can be modulated is poorly explored. Here, we discovered that human SLC25A39, a mitochondrial transporter critical for mitochondrial glutathione uptake, is a short-lived protein under dual regulation at the protein level. Co-immunoprecipitation mass spectrometry and CRISPR knockout (KO) in mammalian cells identified that mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1. SLC25A39 senses mitochondrial iron-sulfur cluster using four matrix cysteine residues and inhibits its degradation. SLC25A39 protein regulation is robust in developing and mature neurons. This dual transporter regulation, by protein quality control and metabolic sensing, allows modulating mitochondrial glutathione level in response to iron homeostasis, opening avenues for exploring regulation of metabolic compartmentalization. Neuronal SLC25A39 regulation connects mitochondrial protein quality control, glutathione, and iron homeostasis, which were previously unrelated biochemical features in neurodegeneration.

LONP1 ameliorates liver injury and improves gluconeogenesis dysfunction in acute-on-chronic liver failure / 中华医学杂志(英文版)

BACKGROUND@#Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown.@*METHODS@#In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF.@*RESULTS@#We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders.@*CONCLUSION@#We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.

Intraneuronal ß-amyloid impaired mitochondrial proteostasis through the impact on LONP1.

Mitochondrial dysfunction plays a critical role in the pathogenesis of Alzheimer's disease (AD). Mitochondrial proteostasis regulated by chaperones and proteases in each compartment of mitochondria is critical for mitochondrial function, and it is suspected that mitochondrial proteostasis deficits may be involved in mitochondrial dysfunction in AD. In this study, we identified LONP1, an ATP-dependent protease in the matrix, as a top Aß42 interacting mitochondrial protein through an unbiased screening and found significantly decreased LONP1 expression and extensive mitochondrial proteostasis deficits in AD experimental models both in vitro and in vivo, as well as in the brain of AD patients. Impaired METTL3-m6A signaling contributed at least in part to Aß42-induced LONP1 reduction. Moreover, Aß42 interaction with LONP1 impaired the assembly and protease activity of LONP1 both in vitro and in vivo. Importantly, LONP1 knockdown caused mitochondrial proteostasis deficits and dysfunction in neurons, while restored expression of LONP1 in neurons expressing intracellular Aß and in the brain of CRND8 APP transgenic mice rescued Aß-induced mitochondrial deficits and cognitive deficits. These results demonstrated a critical role of LONP1 in disturbed mitochondrial proteostasis and mitochondrial dysfunction in AD and revealed a mechanism underlying intracellular Aß42-induced mitochondrial toxicity through its impact on LONP1 and mitochondrial proteostasis.

A Transient π-π or Cation-π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in Escherichia coli.

The Escherichia coli ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr91st residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the degron site (GFIMRP147th) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe143rd residue in degron site is necessary for SulA native folded structure. However, the functional association of this degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpYY91FQ, ClpYY91HQ, and ClpYY91WQ, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced ß-galactosidase activity of cpsB::lacZ, and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulAF143H, SulAF143K and SulAF143W, harboring a ring structure or a cation side-group in 143rd residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 °C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpYY91FQ and ClpYY91HQ, were capable of effectively degrading SulAF143H and SulAF143K, highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient π-π or cation-π interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation.

Exercise combined with postbiotics treatment results in synergistic improvement of mitochondrial function in the brain of male transgenic mice for Alzheimer's disease.

BACKGROUND: It has been suggested that exercise training and postbiotic supplement could decelerate the progress of functional and biochemical deterioration in double transgenic mice overexpresses mutated forms of the genes for human amyloid precursor protein (APPsw) and presenilin 1 (m146L) (APP/PS1TG). Our earlier published data indicated that the mice performed better than controls on the Morris Maze Test parallel with decreased occurrence of amyloid-ß plaques in the hippocampus. We investigated the neuroprotective and therapeutic effects of high-intensity training and postbiotic supplementation. METHODS: Thirty-two adult APP/PS1TG mice were randomly divided into four groups: (1) control, (2) high-intensity training (3) postbiotic, (4) combined (training and postbiotic) treatment for 20 weeks. In this study, the whole hemibrain without hippocampus was used to find molecular traits explaining improved brain function. We applied qualitative RT-PCR for gene expression, Western blot for protein level, and Zymography for LONP1 activity. Disaggregation analysis of Aß-40 was performed in the presence of Lactobacillus acidophilus and Bifidobacterium longum lysate. RESULTS: We found that exercise training decreased Alzheimer's Disease (AD)-related gene expression (NF-kB) that was not affected by postbiotic treatment. The preparation used for postbiotic treatment is composed of tyndallized Bifidobacterium longum and Lactobacillus acidophilus. Both of the postbiotics effectively disaggregated amyloid-ß/Aß-40 aggregates by chelating Zn2+ and Cu2+ ions. The postbiotic treatment decreased endogenous human APPTG protein expression and mouse APP gene expression in the hemibrains. In addition, the postbiotic treatment elevated mitochondrial LONP1 activity as well. CONCLUSION: Our findings revealed distinct mechanisms behind improved memory performance in the whole brain: while exercise training modulates NF-kB signaling pathway regulating immune response until postbiotic diminishes APP gene expression, disaggregates pre-existing amyloid-ß plaques and activates mitochondrial protein quality control in the region of brain out of hippocampus. Using the above treatments complements and efficiently slows down the development of AD.

Integration of FUNDC1-associated mitochondrial protein import and mitochondrial quality control contributes to TDP-43 degradation.

Though TDP-43 protein can be translocated into mitochondria and causes mitochondrial damage in TDP-43 proteinopathy, little is known about how TDP-43 is imported into mitochondria. In addition, whether mitochondrial damage is caused by mitochondrial mislocalization of TDP-43 or a side effect of mitochondria-mediated TDP-43 degradation remains to be investigated. Here, our bioinformatical analyses reveal that mitophagy receptor gene FUNDC1 is co-expressed with TDP-43, and both TDP-43 and FUNDC1 expression is correlated with genes associated with mitochondrial protein import pathway in brain samples of patients diagnosed with TDP-43 proteinopathy. FUNDC1 promotes mitochondrial translocation of TDP-43 possibly by promoting TDP-43-TOM70 and DNAJA2-TOM70 interactions, which is independent of the LC3 interacting region of FUNDC1 in cellular experiments. In the transgenic fly model of TDP-43 proteinopathy, overexpressing FUNDC1 enhances TDP-43 induced mitochondrial damage, whereas down-regulating FUNDC1 reverses TDP-43 induced mitochondrial damage. FUNDC1 regulates mitochondria-mediated TDP-43 degradation not only by regulating mitochondrial TDP-43 import, but also by increasing LONP1 level and by activating mitophagy, which plays important roles in cytosolic TDP-43 clearance. Together, this study not only uncovers the mechanism of mitochondrial TDP-43 import, but also unravels the active role played by mitochondria in regulating TDP-43 homeostasis.

Autoregulatory control of mitochondrial glutathione homeostasis.

Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.

The heat shock protein LarA activates the Lon protease in response to proteotoxic stress.

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.

Determining the 3-substituted Coumarins inhibitory potential against the HslV protease of E. coli.

OBJECTIVE: The growing bacterial resistance towards classical antibiotics demands the development of novel approaches for the effective treatment of potentially fatal bacterial infections in humans. Proteostasis is crucial for the survival of every living cell, as several important physiological functions depend on well-regulated proteostasis. Within bacteria, the regulation of proteostasis relies on AAA+ (Adenosine 5'-triphosphatases associated with diverse cellular activities), ATPases, such as the HslVU complex (heat shock locus gene products U and V), along with other proteases. The HslVU protease/chaperon complex is thought to be the progenitor of the eukaryotic proteasome that regulates proteostasis mostly in prokaryotes. This study aimed to determine the inhibitory potential of 3-substituted coumarin derivatives against Escherichia coli heat shock locus V (HslV) protease. MATERIALS AND METHODS: In this study, twenty-three derivatives of 3-substituted coumarin were assessed for their inhibitory potential against E. coli HslV protease using both in-vitro and in-silico techniques. RESULTS: Among all the tested compounds, US-I-64, US-I-66, US-I-67, and US-I-68 displayed notable inhibitory potential against the HslV protease, showing IC50 (half maximal inhibitory concentration) values ranging from 0.2 to 0.73 µM. Additionally, the inhibitory potential of these compounds against the eukaryotic proteasome was also evaluated using a separate in-silico study. It was found that these compounds did not bind with the proteasomal active site, suggesting no apparent side effects of these lead molecules. CONCLUSIONS: These identified HslV protease inhibitors can be used for the development of novel and safer anti-bacterial drugs.

Mitochondrial oxidative stress regulates LonP1-TDP-43 pathway and rises mitochondrial damage in carbon tetrachloride-induced liver fibrosis.

Carbon tetrachloride (CCl4)-mediated liver damage has been well recognized, but the sources and mechanisms of mitochondrial damage during this progress still remain poorly understood. Accumulating evidence has revealed that LonP1-TDP-43 pathway affect proper mitochondrial integrity and function in neurodegenerative diseases. The current study aims to investigate whether mitochondrial oxidative stress regulate LonP1-TDP-43 pathway and the possible roles of this pathway in CCl4-driven liver fibrosis. We found that TDP-43 interacted with LonP1 in chronic CCl4 exposure-induced hepatic fibrogenesis. Moreover, CCl4 led to deficiency of LonP1 and excessive accumulation of TDP-43 on mitochondria. Particularly, the gene correlation analysis for liver fibrosis patients RNA sequencing (RNA-seq) results (GSE159676) showed an obvious negative correlation between LonP1 and TDP-43. By contrast, MitoQ enhanced the occurrence of mitochondrial unfolded protein response (mtUPR), especially the activation of LonP1 after CCl4 treatment. Importantly, mitochondrial antioxidant also promoted the degradation of TDP-43 and alleviated mitochondrial damage. In addition, our results showed that CCl4 induced the release of mitochondrial DNA (mtDNA) and effectively elevated cGAS-STING-mediated immune response, which can be inhibited by MitoQ. Finally, MitoQ prevented CCl4-induced liver fibrosis. Together, our study revealed that LonP1-TDP-43 pathway mediated by mitochondrial oxidative stress participated in the progress of CCl4-drived liver fibrosis. Therefore, mitigating or reversing mitochondrial damage through targeting LonP1-TDP-43 pathway may serve as a promising therapeutic strategy for CCl4 exposure-induced liver diseases.

Differential sensitivity of the yeast Lon protease Pim1p to impaired mitochondrial respiration.

Mitochondria are essential organelles whose proteome is well protected by regulated protein degradation and quality control. While the ubiquitin-proteasome system can monitor mitochondrial proteins that reside at the mitochondrial outer membrane or are not successfully imported, resident proteases generally act on proteins within mitochondria. Herein, we assess the degradative pathways for mutant forms of three mitochondrial matrix proteins (mas1-1HA, mas2-11HA, and tim44-8HA) in Saccharomyces cerevisiae. The degradation of these proteins is strongly impaired by loss of either the matrix AAA-ATPase (m-AAA) (Afg3p/Yta12p) or Lon (Pim1p) protease. We determine that these mutant proteins are all bona fide Pim1p substrates whose degradation is also blocked in respiratory-deficient "petite" yeast cells, such as in cells lacking m-AAA protease subunits. In contrast, matrix proteins that are substrates of the m-AAA protease are not affected by loss of respiration. The failure to efficiently remove Pim1p substrates in petite cells has no evident relationship to Pim1p maturation, localization, or assembly. However, Pim1p's autoproteolysis is intact, and its overexpression restores substrate degradation, indicating that Pim1p retains some functionality in petite cells. Interestingly, chemical perturbation of mitochondria with oligomycin similarly prevents degradation of Pim1p substrates. Our results demonstrate that Pim1p activity is highly sensitive to mitochondrial perturbations such as loss of respiration or drug treatment in a manner that we do not observe with other proteases.

Excessive ER-phagy contributes to ochratoxin A-induced apoptosis.

The nephrotoxic secondary fungal metabolite ochratoxin A (OTA) is ubiquitously existed in foodstuffs and feeds. Although our earlier research provided preliminary evidence that endoplasmic reticulum (ER) was crucial in OTA-induced nephrotoxicity, more research is necessary to understand the fine-tune mechanisms involving ER stress (ERS), ER-phagy, and apoptosis. In the present study, the cell viability and protein expressions of human proximal tubule epithelial (HK-2) cells in response to OTA and/or chloroquine/rapamycin/sodium phenylbutyrate/tunicamycin were determined via cell viability assay, apoptosis analysis, and Western blot analysis. The findings showed that a 24 h-treatment of 0.25-4 µM OTA could significantly reduced the cell viability (P < 0.05), which notably increased with the addition of chloroquine and sodium phenylbutyrate, while decreased with the addition of rapamycin and tunicamycin as compared to group OTA (P < 0.05). A 24 h-treatment of 1-4 µM OTA could markedly induce apoptosis via increasing the protein expressions of GRP78, p-eIF2α, Chop, LC3B-II, Bak, and Bax, and inhibiting the protein expressions of DDRGK1, UBA5, Lonp1, Tex264, FAM134B, p-mTOR, p62, and Bcl-2 in HK-2 cells (P < 0.05). In conclusion, OTA activated ERS, unfolded protein response, and subsequent excessive ER-phagy, thus inducing apoptosis, and the vicious cycle between excessive ER-phagy and ERS could further promote apoptosis in vitro.

Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism.

Mutations in GBA1, the gene encoding the lysosomal enzyme ß-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.