Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae).

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Plasmodium falciparum proteases as new drug targets with special focus on metalloproteases.

Malaria poses a significant global health threat particularly due to the prevalence of Plasmodium falciparum infection. With the emergence of parasite resistance to existing drugs including the recently discovered artemisinin, ongoing research seeks novel therapeutic avenues within the malaria parasite. Proteases are promising drug targets due to their essential roles in parasite biology, including hemoglobin digestion, merozoite invasion, and egress. While exploring the genomic landscape of Plasmodium falciparum, it has been revealed that there are 92 predicted proteases, with only approximately 14 of them having been characterized. These proteases are further distributed among 26 families grouped into five clans: aspartic proteases, cysteine proteases, metalloproteases, serine proteases, and threonine proteases. Focus on metalloprotease class shows further role in organelle processing for mitochondria and apicoplasts suggesting the potential of metalloproteases as viable drug targets. Holistic understanding of the parasite intricate life cycle and identification of potential drug targets are essential for developing effective therapeutic strategies against malaria and mitigating its devastating global impact.

The mitochondrial protease PARL is required for spermatogenesis.

Mitochondrial function plays an important role in the maintenance of male fertility. However, the mechanisms underlying mitochondrial defect-related infertility remain mostly unclear. Here we show that a deficiency of PARL (Parl-/-), a mitochondrial protease, causes complete arrest of spermatogenesis during meiosis I. PARL deficiency led to severe downregulation of proteins of respiratory chain complex IV in testes that did not occur in other tested organs, causing a deficit in complex IV activity and ATP production. Furthermore, Parl-/- testes showed an almost complete loss of HSD17B3, a protein of the sER responsible for the last step in testosterone synthesis. While testosterone production appeared to be restored by overexpression of HSD17B12, loss of the canonical testosterone synthesis led to an upregulation of luteinizing hormone (LH) and of LH-regulated responses. These results suggest an important impact of the downstream regulation of mitochondrial defects that manifest in a cell-type-specific manner and extend beyond mitochondria.

Structure of the Borrelia burgdorferi ATP-dependent metalloprotease FtsH in its functionally relevant hexameric form.

ATP-dependent proteases FtsH are conserved in bacteria, mitochondria, and chloroplasts, where they play an essential role in degradation of misfolded/unneeded membrane and cytosolic proteins. It has also been demonstrated that the FtsH homologous protein BB0789 is crucial for mouse and tick infectivity and in vitro growth of the Lyme disease-causing agent Borrelia burgdorferi. This is not surprising, considering B. burgdorferi complex life cycle, residing in both in mammals and ticks, which requires a wide range of membrane proteins and short-lived cytosolic regulatory proteins to invade and persist in the host organism. In the current study, we have solved the crystal structure of the cytosolic BB0789166-614, lacking both N-terminal transmembrane α-helices and the small periplasmic domain. The structure revealed the arrangement of the AAA+ ATPase and the zinc-dependent metalloprotease domains in a hexamer ring, which is essential for ATPase and proteolytic activity. The AAA+ domain was found in an ADP-bound state, while the protease domain showed coordination of a zinc ion by two histidine residues and one aspartic acid residue. The loop region that forms the central pore in the oligomer was poorly defined in the crystal structure and therefore predicted by AlphaFold to complement the missing structural details, providing a complete picture of the functionally relevant hexameric form of BB0789. We confirmed that BB0789 is functionally active, possessing both protease and ATPase activities, thus providing novel structural-functional insights into the protein, which is known to be absolutely necessary for B. burgdorferi to survive and cause Lyme disease.

The cyanobacterial FtsH4 protease controls accumulation of protein factors involved in the biogenesis of photosystem I.

Membrane-bound FtsH proteases are universally present in prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. These metalloproteases are often critical for viability and play both protease and chaperone roles to maintain cellular homeostasis. In contrast to most bacteria bearing a single ftsH gene, cyanobacteria typically possess four FtsH proteases (FtsH1-4) forming heteromeric (FtsH1/3 and FtsH2/3) and homomeric (FtsH4) complexes. The functions and substrate repertoire of each complex are however poorly understood. To identify substrates of the FtsH4 protease complex we established a trapping assay in the cyanobacterium Synechocystis PCC 6803 utilizing a proteolytically inactivated trapFtsH4-His. Around 40 proteins were specifically enriched in trapFtsH4 pulldown when compared with the active FtsH4. As the list of putative FtsH4 substrates contained Ycf4 and Ycf37 assembly factors of Photosystem I (PSI), its core PsaB subunit and the IsiA chlorophyll-binding protein that associates with PSI during iron stress, we focused on these PSI-related proteins. Therefore, we analysed their degradation by FtsH4 in vivo in Synechocystis mutants and in vitro using purified substrates. The data confirmed that FtsH4 degrades Ycf4, Ycf37, IsiA, and also the individual PsaA and PsaB subunits in the unassembled state but not when assembled within the PSI complexes. A possible role of FtsH4 in the PSI life-cycle is discussed.

Disruption of Mitophagy Flux through the PARL-PINK1 Pathway by CHCHD10 Mutations or CHCHD10 Depletion.

Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS-FTD patients, and the CHCHD10S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10WT) normally enhances these measures. Specifically, we show that CHCHD10R15L and CHCHD10S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL-PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10-PARL-PINK1 pathway.

Co-option of an Astacin Metalloprotease Is Associated with an Evolutionarily Novel Feeding Morphology in a Predatory Nematode.

Animals consume a wide variety of food sources to adapt to different environments. However, the genetic mechanisms underlying the acquisition of evolutionarily novel feeding morphology remain largely unknown. While the nematode Caenorhabditis elegans feeds on bacteria, the satellite species Pristionchus pacificus exhibits predatory feeding behavior toward other nematodes, which is an evolutionarily novel feeding habit. Here, we found that the astacin metalloprotease Ppa-NAS-6 is required for the predatory killing by P. pacificus. Ppa-nas-6 mutants were defective in predation-associated characteristics, specifically the tooth morphogenesis and tooth movement during predation. Comparison of expression patterns and rescue experiments of nas-6 in P. pacificus and C. elegans suggested that alteration of the spatial expression patterns of NAS-6 may be vital for acquiring predation-related traits. Reporter analysis of the Ppa-nas-6 promoter in C. elegans revealed that the alteration in expression patterns was caused by evolutionary changes in cis- and trans-regulatory elements. This study suggests that the co-option of a metalloprotease is involved in an evolutionarily novel feeding morphology.

Loss of UCP2 causes mitochondrial fragmentation by OMA1-dependent proteolytic processing of OPA1 in podocytes.

Mitochondrial dysfunction plays an important role in the onset and progression of podocyte injury and proteinuria. However, the process by which the change in the podocyte mitochondria occurs is not well understood. Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which is located in the mitochondrial inner membrane. Here, we reported that mice with podocyte-specific Ucp2 deficiency developed podocytopathy with proteinuria with aging. Furthermore, those mice exhibited increased proteinuria in experimental models evoked by Adriamycin. Our findings suggest that UCP2 mediates mitochondrial dysfunction by regulating mitochondrial dynamic balance. Ucp2-deleted podocytes exhibited increased mitochondrial fission and deficient in ATP production. Mechanistically, opacity protein 1 (OPA1), a key protein in fusion of mitochondrial inner membrane, was regulated by UCP2. Ucp2 deficiency promoted proteolysis of OPA1 by activation OMA1 which belongs to mitochondrial inner membrane zinc metalloprotease. Those finding demonstrate the role of UCP2 in mitochondrial dynamics in podocytes and provide new insights into pathogenesis associated with podocyte injury and proteinuria.

Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia.

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

Removal of extracellular human amyloid beta aggregates by extracellular proteases in C. elegans.

The amyloid beta (Aß) plaques found in Alzheimer's disease (AD) patients' brains contain collagens and are embedded extracellularly. Several collagens have been proposed to influence Aß aggregate formation, yet their role in clearance is unknown. To investigate the potential role of collagens in forming and clearance of extracellular aggregates in vivo, we created a transgenic Caenorhabditis elegans strain that expresses and secretes human Aß1-42. This secreted Aß forms aggregates in two distinct places within the extracellular matrix. In a screen for extracellular human Aß aggregation regulators, we identified different collagens to ameliorate or potentiate Aß aggregation. We show that a disintegrin and metalloprotease a disintegrin and metalloprotease 2 (ADM-2), an ortholog of ADAM9, reduces the load of extracellular Aß aggregates. ADM-2 is required and sufficient to remove the extracellular Aß aggregates. Thus, we provide in vivo evidence of collagens essential for aggregate formation and metalloprotease participating in extracellular Aß aggregate removal.

Investigating the association between serum ADAM/ADAMTS levels and bone mineral density by mendelian randomization study.

PURPOSE: A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motif (ADAMTS) have been reported potentially involved in bone metabolism and related to bone mineral density. This Mendelian Randomization (MR) analysis was performed to determine whether there are causal associations of serum ADAM/ADAMTS with BMD in rid of confounders. METHODS: The genome-wide summary statistics of four site-specific BMD measurements were obtained from studies in individuals of European ancestry, including forearm (n = 8,143), femoral neck (n = 32,735), lumbar spine (n = 28,498) and heel (n = 426,824). The genetic instrumental variables for circulating levels of ADAM12, ADAM19, ADAM23, ADAMTS5 and ADAMTS6 were retrieved from the latest genome-wide association study of European ancestry (n = 5336 ~ 5367). The estimated causal effect was given by the Wald ratio for each variant, the inverse-variance weighted model was used as the primary approach to combine estimates from multiple instruments, and sensitivity analyses were conducted to assess the robustness of MR results. The Bonferroni-corrected significance was set at P < 0.0025 to account for multiple testing, and a lenient threshold P < 0.05 was considered to suggest a causal relationship. RESULTS: The causal effects of genetically predicted serum ADAM/ADAMTS levels on BMD measurements at forearm, femoral neck and lumbar spine were not statistically supported by MR analyses. Although causal effect of ADAMTS5 on heel BMD given by the primary MR analysis (ß = -0.006, -0.010 to 0.002, P = 0.004) failed to reach Bonferroni-corrected significance, additional MR approaches and sensitivity analyses indicated a robust causal relationship. CONCLUSION: Our study provided suggestive evidence for the causal effect of higher serum levels of ADAMTS5 on decreased heel BMD, while there was no supportive evidence for the associations of ADAM12, ADAM19, ADAM23, and ADAMTS6 with BMD at forearm, femoral neck and lumbar spine in Europeans.

Mitochondrial defects caused by PARL deficiency lead to arrested spermatogenesis and ferroptosis.

Impaired spermatogenesis and male infertility are common manifestations associated with mitochondrial diseases, yet the underlying mechanisms linking these conditions remain elusive. In this study, we demonstrate that mice deficient for the mitochondrial intra-membrane rhomboid protease PARL, a recently reported model of the mitochondrial encephalopathy Leigh syndrome, develop early testicular atrophy caused by a complete arrest of spermatogenesis during meiotic prophase I, followed by degeneration and death of arrested spermatocytes. This process is independent of neurodegeneration. Interestingly, genetic modifications of PINK1, PGAM5, and TTC19 - three major substrates of PARL with important roles in mitochondrial homeostasis - fail to reproduce or modify this severe phenotype, indicating that the spermatogenic arrest arises from distinct molecular pathways. We further observed severe abnormalities in mitochondrial ultrastructure in PARL-deficient spermatocytes, along with prominent electron transfer chain defects, disrupted coenzyme Q (CoQ) biosynthesis, and metabolic rewiring. These mitochondrial defects are associated with a germ cell-specific decrease in GPX4 expression leading arrested spermatocytes to ferroptosis - a regulated cell death modality characterized by uncontrolled lipid peroxidation. Our results suggest that mitochondrial defects induced by PARL depletion act as an initiating trigger for ferroptosis in primary spermatocytes through simultaneous effects on GPX4 and CoQ - two major inhibitors of ferroptosis. These findings shed new light on the potential role of ferroptosis in the pathogenesis of mitochondrial diseases and male infertility warranting further investigation.

Up to 9% of men are thought to experience infertility. These individuals may not produce enough healthy sperm cells. The root cause of infertility is often not discovered but, in some cases, it is associated with genetic defects in cell compartments known as mitochondria. Mitochondria are responsible for converting energy from food into a form of chemical energy cells need to power vital processes. However, it remains unclear how defects in mitochondria contribute to male infertility. Leigh syndrome is one of the most prevalent and severe diseases caused by genetic defects in mitochondria. The condition often develops in childhood and affects the nervous system, muscle and other organs, leading to many symptoms including muscle weakness and neurological regression. A previous study found that mutant mice that lack an enzyme, called PARL, display symptoms that are similar to those observed in humans with Leigh syndrome. PARL is found inside mitochondria where it cuts specific proteins to ensure they are working correctly in the cells. Radaelli et al. used extensive microscopy and biochemical analyses to study the fertility of male mice lacking PARL. The experiments revealed that the males were infertile due to a failure to produce sperm: spermatocytes, which usually develop into sperm cells, where much more likely to die in mice without PARL (by a process known as ferroptosis). Further experiments demonstrated that the mitochondria of the mutant mice had a shortage of two crucial molecules, a protein called GPX4 and a lipid called Coenzyme Q, which are required to prevent death by ferroptosis. It appears that this shortage was responsible for the demise of spermatocytes in the male mutant mice affected by infertility. These findings reveal a new role for PARL in the body and provide evidence that mitochondrial defects in living mammals can trigger ferroptosis, thereby contributing to male infertility. In the future, this research may pave the way for new treatments for male infertility and other diseases associated with defects in mitochondria.

The Venom Composition of the Snake Tribe Philodryadini: 'Omic' Techniques Reveal Intergeneric Variability among South American Racers.

Snakes of the Philodryadini tribe are included in the Dipsadidae family, which is a diverse group of rear-fanged snakes widespread in different ecological conditions, including habitats and diet. However, little is known about the composition and effects of their venoms despite their relevance for understanding the evolution of these snakes or even their impact on the occasional cases of human envenoming. In this study, we integrated venom gland transcriptomics, venom proteomics and functional assays to characterize the venoms from eight species of the Philodryadini tribe, which includes the genus Philodryas, Chlorosoma and Xenoxybelis. The most abundant components identified in the venoms were snake venom metalloproteinases (SVMPs), cysteine-rich secretory proteins (CRISPs), C-type lectins (CTLs), snake endogenous matrix metalloproteinases type 9 (seMMP-9) and snake venom serinoproteinases (SVSPs). These protein families showed a variable expression profile in each genus. SVMPs were the most abundant components in Philodryas, while seMMP-9 and CRISPs were the most expressed in Chlorosoma and Xenoxybelis, respectively. Lineage-specific differences in venom composition were also observed among Philodryas species, whereas P. olfersii presented the highest amount of SVSPs and P. agassizii was the only species to express significant amounts of 3FTx. The variability observed in venom composition was confirmed by the venom functional assays. Philodryas species presented the highest SVMP activity, whereas Chlorosoma species showed higher levels of gelatin activity, which may correlate to the seMMP-9 enzymes. The variability observed in the composition of these venoms may be related to the tribe phylogeny and influenced by their diets. In the presented study, we expanded the set of venomics studies of the Philodryadini tribe, which paves new roads for further studies on the evolution and ecology of Dipsadidae snakes.

Bi-allelic variants in ASTL cause abnormal fertilization or oocyte maturation defects.

Fertilization is a fundamental process of development, and the blocking mechanisms act at the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, permeating and fusing after fertilization. In clinical practice, some couples undergoing recurrent IVF failures that mature oocytes had abnormal fertilization for unknown reason. Ovastacin encoded by ASTL cleave the ZP protein ZP2 and play a key role in preventing polyspermy. Here, we identified bi-allelic variants in ASTL that are mainly characterized by fertilization problems in humans. All four independent affected individuals had bi-allelic frameshift variants or predicted damaging missense variants, which follow a Mendelian recessive inheritance pattern. The frameshift variants significantly decreased the quantity of ASTL protein in vitro. And all missense variants affected the enzymatic activity that cleaves ZP2 in mouse egg in vitro. Three knock-in female mice (corresponding to three missense variants in patients) all show subfertility due to low embryo developmental potential. This work presents strong evidence that pathogenic variants in ASTL cause female infertility and provides a new genetic marker for the diagnosis of fertilization problems.

An OMA1 redox site controls mitochondrial homeostasis, sarcoma growth, and immunogenicity.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell-deficient, mice. These cells prime CD8+ lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.

NMR structure of emfourin, a novel protein metalloprotease inhibitor: Insights into the mechanism of action.

Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.

Chloroplast metalloproteinase SlL2 reduces the thermotolerance of tomato by decreasing the content of SlCDJ1.

Chloroplast is one of the most sensitive organelles to heat stress in plants. In chloroplasts, various proteases affect photosynthesis by degrading proteins under stress conditions. Tomato Lutescent2 (SlL2), a chloroplast zinc metalloprotease, was previously reported to alter chloroplast development and delay fruit ripening. However, its enzyme activity and roles in plant response to abiotic stress are still unclear. Here, we confirmed that the SlL2 protein which localized on thylakoid membrane was an ATP-independent hydrolase, and SlL2 gene responded to heat stress. Phenotype analysis showed that SlL2 plays a negative role in the heat-response mechanism. Under heat stress, the transgenic plants overexpressing SlL2 (OE) grew worse than the wild type (WT), as reflected by their decreased membrane stability, osmotic-regulating substance, and antioxidative enzyme activities, as well as increased reactive oxygen species (ROS) accumulation. By contrast, l2 mutant line showed the opposite phenotype and corresponding physiological indices under heat stress. In addition, overexpression of SlL2 decreased the photosynthetic activities, especially photosystem II. Moreover, SlL2 was found to interact with chloroplast-located chaperone protein SlCDJ1, decreasing its content under heat stress. These results indicate that SlL2 reduces the thermotolerance of tomato by reducing the content of SlCDJ1.

Chemical Blockage of the Mitochondrial Rhomboid Protease PARL by Novel Ketoamide Inhibitors Reveals Its Role in PINK1/Parkin-Dependent Mitophagy.

The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.