Mitochondrial membrane potential and oxidative stress interact to regulate Oma1-dependent processing of Opa1 and mitochondrial dynamics.

Mitochondrial form and function are regulated by the opposing forces of mitochondrial dynamics: fission and fusion. Mitochondrial dynamics are highly active and consequential during neuronal ischemia/reperfusion (I/R) injury. Mitochondrial fusion is executed at the mitochondrial inner membrane by Opa1. The balance of long (L-Opa1) and proteolytically cleaved short (S-Opa1) isoforms is critical for efficient fusion. Oma1 is the predominant stress-responsive protease for Opa1 processing. In neuronal cell models, we assessed Oma1 and Opa1 regulation during mitochondrial stress. In an immortalized mouse hippocampal neuron line (HT22), Oma1 was sensitive to mitochondrial membrane potential depolarization (rotenone, FCCP) and hyperpolarization (oligomycin). Further, oxidative stress was sufficient to increase Oma1 activity and necessary for depolarization-induced proteolysis. We generated Oma1 knockout (KO) HT22 cells that displayed normal mitochondrial morphology and fusion capabilities. FCCP-induced mitochondrial fragmentation was exacerbated in Oma1 KO cells. However, Oma1 KO cells were better equipped to perform restorative fusion after fragmentation, presumably due to preserved L-Opa1. We extended our investigations to a combinatorial stress of neuronal oxygen-glucose deprivation and reoxygenation (OGD/R), where we found that Opa1 processing and Oma1 activation were initiated during OGD in an ROS-dependent manner. These findings highlight a novel dependence of Oma1 on oxidative stress in response to depolarization. Further, we demonstrate contrasting fission/fusion roles for Oma1 in the acute response and recovery stages of mitochondrial stress. Collectively, our results add intersectionality and nuance to the previously proposed models of Oma1 activity.

Caffeic Acid Phenethyl Ester Administration Reduces Enterotoxigenic Bacteroides fragilis-Induced Colitis and Tumorigenesis.

The human colonic commensal enterotoxigenic Bacteroides fragilis (ETBF) is associated with chronic colitis and colon cancer. ETBF colonization induces colitis via the Bacteroides fragilis toxin (BFT). BFT secreted by ETBF cause colon inflammation via E-cadherin cleavage/NF-κB signaling. ETBF promotes colon tumorigenesis via interleukin 17A (IL-17A)/CXCL-dependent inflammation, but its bioactive therapeutics in ETBF-promoted tumorigenesis remain unexplored. In the current study, we investigated the caffeic acid phenethyl ester (CAPE) in the murine model of ETBF colitis and tumorigenesis. In this study, we observed that CAPE treatment mitigated inflammation induced by ETBF in mice. Additionally, our findings indicate that CAPE treatment offers protective effects against ETBF-enhanced colon tumorigenesis in a mouse model of colitis-associated colon cancer induced by azoxymethane (AOM) and dextran sulfate sodium. Notably, the decrease in colon tumorigenesis following CAPE administration correlates with a reduction in the expression of IL-17A and CXCL1 in the gastrointestinal tract. The molecular mechanism for CAPE-induced protection against ETBF-mediated tumorigenesis is mediated by IL-17A/CXCL1, and by NF-κB activity in intestinal epithelial cells. Our findings indicate that CAPE may serve as a preventive agent against the development of ETBF-induced colitis and colorectal cancer (CRC).

Structural basis of divergent substrate recognition and inhibition of human neurolysin.

A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1-13) potently inhibits the Nln activity while dynorphin A (1-8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.

Mitochondrial metallopeptidase OMA1 in cancer.

Our understanding of the roles that mitochondria play in cellular physiology has evolved drastically-from a mere cellular energy supplier to a crucial regulator of metabolic and signaling processes, particularly in the context of development and progression of human diseases such as cancers. The present review examines the role of OMA1, a conserved, redox-sensitive metallopeptidase in cancer biology. OMA1's involvement in mitochondrial quality control, redox activity, and stress responses underscores its potential as a novel target in cancer diagnosis and treatment. However, our incomplete understanding of OMA1's regulation and structural detail presents ongoing challenges to target OMA1 for therapeutic purposes. Further exploration of OMA1 holds promise in uncovering novel insights into cancer mechanisms and therapeutic strategies. In this chapter, we briefly summarize our current knowledge about OMA1, its redox-regulation, and emerging role in certain cancers.

Insight into the role of Streptococcus suis zinc metalloprotease C from the new serotype causing meningitis in piglets.

Streptococcus suis (S. suis) is an important gram-positive pathogen and an emerging zoonotic pathogen that causes meningitis in swine and humans. Although several virulence factors have been characterized in S. suis, the underlying mechanisms of pathogenesis are not fully understood. In this study, we identified Zinc metalloproteinase C (ZmpC) probably as a critical virulence factor widely distributed in S. suis strains. ZmpC was identified as a critical facilitator in the development of bacterial meningitis, as evidenced by the detection of increased expression of TNF-α, IL-8, and matrix metalloprotease 9 (MMP-9). Subcellular localization analysis further revealed that ZmpC was localized to the cell wall surface and gelatin zymography analysis showed that ZmpC could cleave human MMP-9. Mice challenge demonstrated that ZmpC provided protection against S. suis CZ130302 (serotype Chz) and ZY05719 (serotype 2) infection. In conclusion, these results reveal that ZmpC plays an important role in promoting CZ130302 to cause mouse meningitis and may be a potential candidate for a S. suis CZ130302 vaccine.

Mutations in fibulin-1 and collagen IV suppress the short healthspan of mig-17/ADAMTS mutants in Caenorhabditis elegans.

The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloprotease MIG-17 plays a crucial role in the migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans. MIG-17 is secreted from the body wall muscle cells and localizes to the basement membranes (BMs) of various tissues including the gonadal BM where it regulates DTC migration through its catalytic activity. Missense mutations in the BM protein genes, let-2/collagen IV a2 and fbl-1/fibulin-1, have been identified as suppressors of the gonadal defects observed in mig-17 mutants. Genetic analyses indicate that LET-2 and FBL-1 act downstream of MIG-17 to regulate DTC migration. In addition to the control of DTC migration, MIG-17 also plays a role in healthspan, but not in lifespan. Here, we examined whether let-2 and fbl-1 alleles can suppress the age-related phenotypes of mig-17 mutants. let-2(k196) fully and fbl-1(k201) partly, but not let-2(k193) and fbl-1(k206), suppressed the senescence defects of mig-17. Interestingly, fbl-1(k206), but not fbl-1(k201) or let-2 alleles, exhibited an extended lifespan compared to the wild type when combined with mig-17. These results reveal allele specific interactions between let-2 or fbl-1 and mig-17 in age-related phenotypes, indicating that basement membrane physiology plays an important role in organismal aging.

Split intein-mediated backbone cyclization enhances the stability and activity of staphylokinase, a potent fibrin-selective plasminogen activator.

Staphylokinase (Sak), a small 15 kDa globular protein that is secreted by certain strains of Staphylococcus aureus, shows a potent fibrin-selective thrombolytic activity. Earlier work has shown that Sak could potentially become a low-cost alternative to currently used thrombolytic agents, such as tissue plasminogen activator (tPA). In attempts to improve its potential for clinical applications, numerous modifications of Sak have already been investigated. Here, we have characterized a novel Sak modification, cyclized Sak (cyc-Sak), which was prepared through split-intein mediated protein backbone cyclization. We have characterized the structure, stability and the activity of cyc-Sak using biophysical techniques, limited proteolysis studies and plasminogen (PG)-activation assays. Our results show that cyc-Sak possesses an identical structure, enhanced stability, resistance to proteolysis by exoproteases and improved PG-activation properties compared to its linear counterpart. It can be over-expressed with high yield in the cytoplasm of Escherichia coli and is easily purified in a two-step process. The intein-mediated cyclization occurs spontaneously in vivo during protein expression and does not necessitate further modification steps after purification of the protein. Furthermore, covalent Sak cyclization could be readily combined with other Sak modifications previously proposed, to generate an effective thrombolytic agent with lower immunogenicity and improved stability and activity.

Targeting CK2 eliminates senescent cells and prolongs lifespan in Zmpste24-deficient mice.

Senescent cell clearance is emerging as a promising strategy for treating age-related diseases. Senolytics are small molecules that promote the clearance of senescent cells; however, senolytics are uncommon and their underlying mechanisms remain largely unknown. Here, we investigated whether genomic instability is a potential target for senolytic. We screened small-molecule kinase inhibitors involved in the DNA damage response (DDR) in Zmpste24-/- mouse embryonic fibroblasts, a progeroid model characterized with impaired DDR and DNA repair. 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), which specifically inhibits casein kinase 2 (CK2), was selected and discovered to preferentially trigger apoptosis in Zmpste24-/- cells. Mechanistically, inhibition of CK2 abolished the phosphorylation of heterochromatin protein 1α (HP1α), which retarded the dynamic HP1α dissociation from repressive histone mark H3K9me3 and its relocalization with γH2AX to DNA damage sites, suggesting that disrupting heterochromatin remodeling in the initiation of DDR accelerates apoptosis in senescent cells. Furthermore, feeding Zmpste24-deficient mice with TBB alleviated progeroid features and extended their lifespan. Our study identified TBB as a new class senolytic compound that can reduce age-related symptoms and prolong lifespan in progeroid mice.

Calcium-induced structural transitions are central to the folding, function, and processing of serratiopeptidase zymogen into mature form.

Serratia marcescens is an emerging health-threatening, gram-negative opportunistic pathogen associated with a wide variety of localized and life-threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2-kDa repeats-in-toxin (RTX) family broad-specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram-negative bacteria that exhibit calcium-dependent structural dynamicity and are secreted through a common type-1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C-terminal RTX domain acts as a folding switch, triggering a disordered-to-ordered transition in the enzyme's conformation. Further, the auto-processing of the 16-amino acid N-terminal pro-peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium-rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo-form required by S. marcescens to establish infections and survive in different environmental niches.

Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

Doxycycline decelerates aging in progeria mice.

Beyond the antimicrobial activity, doxycycline (DOX) exhibits longevity-promoting effect in nematodes, while its effect on mammals is unclear. Here, we applied a mouse model of Hutchinson-Gilford progeria syndrome (HGPS), Zmpste24 knockout (KO) mice, and analyzed the antiaging effect of DOX. We found that the DOX treatment prolongs lifespan and ameliorates progeroid features of Zmpste24 KO mice, including the decline of body and tissue weight, exercise capacity and cortical bone density, and the shortened colon length. DOX treatment alleviates the abnormal nuclear envelope in multiple tissues, and attenuates cellular senescence and cell death of Zmpste24 KO and HGPS fibroblasts. DOX downregulates the level of proinflammatory IL6 in both serum and tissues. Moreover, the elevated α-tubulin (K40) acetylation mediated by NAT10 in progeria, is rescued by DOX treatment in the aorta tissues in Zmpste24 KO mice and fibroblasts. Collectively, our study uncovers that DOX can decelerate aging in progeria mice via counteracting IL6 expression and NAT10-mediated acetylation of α-tubulin.

GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

Platelet Membrane-Coated r-SAK Improves Thrombolytic Efficacy by Targeting Thrombus.

Thrombolytic therapy is one of the most effective treatments for thrombus dissolution and recanalization of blocked vessels in thrombotic diseases. However, the application of the thrombolytic strategy has been limited due to unsatisfactory thrombolytic efficacy, relatively higher bleeding complications, and consequently restricted indications. Recombinant staphylokinase (r-SAK) is a third-generation thrombolytic agent produced by genetic engineering technology, which exhibits a better thrombolytic efficacy than urokinase and recombinant streptokinase. Inspired by the natural affinity of platelets in hemostasis and pathological thrombosis, we developed a platelet membrane (PM)-coated r-SAK (PM-r-SAK). Results from animal experiments and human in vitro studies showed that the PM-r-SAK had a thrombolytic efficacy equal to or better than its 4-fold dose of r-SAK. In a totally occluded rabbit femoral artery thrombosis model, the PM-r-SAK significantly shortened the initial recanalization time compared to the same dose and 4-fold dose of r-SAK. Regarding the recanalized vessels, the PM-r-SAK prolonged the time of reperfusion compared to the same dose and 4-fold dose of r-SAK, though the differences were not significant. An in vitro thrombolytic experiment demonstrated that the thrombolytic efficacy of PM-r-SAK could be inhibited by platelet-poor plasma from patients taking aspirin and ticagrelor. PM coating significantly improves the thrombolytic efficacy of r-SAK, which is related to the thrombus-targeting activity of the PM-r-SAK and can be inhibited by aspirin- and ticagrelor-treated plasma.

Nardilysin in vascular smooth muscle cells controls blood pressure via the regulation of calcium dynamics.

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cell (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.

OMA1-Mediated Mitochondrial Dynamics Balance Organellar Homeostasis Upstream of Cellular Stress Responses.

In response to cellular metabolic and signaling cues, the mitochondrial network employs distinct sets of membrane-shaping factors to dynamically modulate organellar structures through a balance of fission and fusion. While these organellar dynamics mediate mitochondrial structure/function homeostasis, they also directly impact critical cell-wide signaling pathways such as apoptosis, autophagy, and the integrated stress response (ISR). Mitochondrial fission is driven by the recruitment of the cytosolic dynamin-related protein-1 (DRP1), while fusion is carried out by mitofusins 1 and 2 (in the outer membrane) and optic atrophy-1 (OPA1) in the inner membrane. This dynamic balance is highly sensitive to cellular stress; when the transmembrane potential across the inner membrane (Δψm) is lost, fusion-active OPA1 is cleaved by the overlapping activity with m-AAA protease-1 (OMA1 metalloprotease, disrupting mitochondrial fusion and leaving dynamin-related protein-1 (DRP1)-mediated fission unopposed, thus causing the collapse of the mitochondrial network to a fragmented state. OMA1 is a unique regulator of stress-sensitive homeostatic mitochondrial balance, acting as a key upstream sensor capable of priming the cell for apoptosis, autophagy, or ISR signaling cascades. Recent evidence indicates that higher-order macromolecular associations within the mitochondrial inner membrane allow these specialized domains to mediate crucial organellar functionalities.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization.

Most mitochondrial proteins originate from the cytosol and require transport into the organelle. Such precursor proteins must be unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. We developed the cell line-based translocase clogging model to discover molecular mechanisms that resolve failed import events in humans. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by VCP/p97 and the proteasome.

YME1L-mediated mitophagy protects renal tubular cells against cellular senescence under diabetic conditions.

BACKGROUND: The senescence of renal tubular epithelial cells (RTECs) is crucial in the progression of diabetic kidney disease (DKD). Accumulating evidence suggests a close association between insufficient mitophagy and RTEC senescence. Yeast mitochondrial escape 1-like 1 (YME1L), an inner mitochondrial membrane metalloprotease, maintains mitochondrial integrity. Its functions in DKD remain unclear. Here, we investigated whether YME1L can prevent the progression of DKD by regulating mitophagy and cellular senescence. METHODS: We analyzed YME1L expression in renal tubules of DKD patients and mice, explored transcriptomic changes associated with YME1L overexpression in RTECs, and assessed its impact on RTEC senescence and renal dysfunction using an HFD/STZ-induced DKD mouse model. Tubule-specific overexpression of YME1L was achieved through the use of recombinant adeno-associated virus 2/9 (rAAV 2/9). We conducted both in vivo and in vitro experiments to evaluate the effects of YME1L overexpression on mitophagy and mitochondrial function. Furthermore, we performed LC-MS/MS analysis to identify potential protein interactions involving YME1L and elucidate the underlying mechanisms. RESULTS: Our findings revealed a significant decrease in YME1L expression in the renal tubules of DKD patients and mice. However, tubule-specific overexpression of YME1L significantly alleviated RTEC senescence and renal dysfunction in the HFD/STZ-induced DKD mouse model. Moreover, YME1L overexpression exhibited positive effects on enhancing mitophagy and improving mitochondrial function both in vivo and in vitro. Mechanistically, our LC-MS/MS analysis uncovered a crucial mitophagy receptor, BCL2-like 13 (BCL2L13), as an interacting partner of YME1L. Furthermore, YME1L was found to promote the phosphorylation of BCL2L13, highlighting its role in regulating mitophagy. CONCLUSIONS: This study provides compelling evidence that YME1L plays a critical role in protecting RTECs from cellular senescence and impeding the progression of DKD. Overexpression of YME1L demonstrated significant therapeutic potential by ameliorating both RTEC senescence and renal dysfunction in the DKD mice. Moreover, our findings indicate that YME1L enhances mitophagy and improves mitochondrial function, potentially through its interaction with BCL2L13 and subsequent phosphorylation. These novel insights into the protective mechanisms of YME1L offer a promising strategy for developing therapies targeting DKD.

Endogenous chondroitin extends the lifespan and healthspan in C. elegans.

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans. However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans. Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.