Bone is a dynamic tissue that is continuously remodeled by tightly-coordinated actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Uncontrolled osteoclastogenesis has a severe impact on skeletal health and thus represents an important therapeutic target in osteolytic diseases. VprBP is a recently identified kinase and plays an important role in stimulating osteoclast differentiation and bone resorption through histone H2AT120 phosphorylation (H2AT120p). We previously developed B32B3 as the first VprBP inhibitor, but its high IC50 value has been a major challenge in using it for osteoclast-triggered bone diseases. In this study, we designed and characterized a series of small molecule compounds derived from B32B3 to identify a novel second-generation inhibitor of VprBP. Our initial screening of B32B3 derivatives identified three compounds, designated as B1486, B3703, and B6756, that are effective in interfering with VprBP kinase activity toward H2AT120p and RANKL-induced differentiation of osteoclast precursor cells (OCPs). However, when their cellular efficacy was systematically accessed over different concentration ranges, B1486 was more potent than B3703 and B6756 at hindering VprBP function in the process of OCP cell differentiation. Consistent with these in vitro findings, B1486 showed significantly improved inhibitory activity against VprBP-driven bone resorption and low bone mass phenotypes in our preclinical studies using zebrafish and mouse models. Taken together, our results indicate that B1486 is a highly potent inhibitor for blocking VprBP-mediated H2AT120p and osteoclastogenic gene silencing, as well as for overcoming osteoporosis generated by excessive osteoclast differentiation.
Natural killer (NK) cells are innate lymphocytes that directly eliminate tumor and virus-infected cells by integrating signals from activating and inhibitory receptors, and their effector functions are tightly coupled to cellular metabolism. Given that the inhibitory receptor PD-1 reprograms T cell metabolism to shape functional fate, the bioenergetic consequences of inhibitory receptor engagement on human NK cells remain largely unexplored, particularly for sialic acid-binding immunoglobulin-like lectin (Siglec-7), a glyco-immune checkpoint receptor. Here, we investigated metabolic programs and effector functions associated with Siglec-7 expression and antibody-mediated Siglec-7 ligation in primary NK cells and NK-92MI cells. Siglec-7POS NK cells exhibited selectively impaired CD107a degranulation under glycolytic and oxidative phosphorylation inhibition, whereas Siglec-7NEG cells remained relatively resistant, indicating distinct energetic wiring between these subsets. Engagement of Siglec-7 by an agonistic antibody induced mitochondrial fission with altered Drp1 phosphorylation, transient mitochondrial depolarization, and broadly suppressed mitochondrial respiration, while concurrently enhancing glycolytic capacity, consistent with a dual metabolic shift upon Siglec-7 ligation. In contrast, sustained Siglec-7 expression in NK-92MI-S cells was associated with globally enhanced mitochondrial respiratory capacity, indicating that sustained Siglec-7 expression and short-term treatment with an agonistic anti-Siglec-7 antibody were associated with distinct metabolic profiles in NK cells. Furthermore, Siglec-7POS NK cells showed increased accumulation of autophagic vacuole, reduced proliferation, and heightened apoptotic susceptibility compared with Siglec-7NEG counterparts. Collectively, these findings support an association between Siglec-7 status, mitochondrial homeostasis, and metabolic fitness in NK cells, with Siglec-7NEG cells retaining a metabolically robust, cytotoxic phenotype.
Chronic inflammation drives many diseases, including cancer, where inflammation is associated with metastasis, the cause of death in 90% of cancer fatalities. Tumor inflammation drives inducible nitric oxide synthase (NOS2) and cyclooxygenase 2 (COX2) expression, each of which is associated with poor outcomes in cancer. Here, we knocked out the NOS2 gene in the murine triple-negative breast cancer allograft tumor model 4T1 and examined metastatic spread from primary mammary tumors to lung in BALB/c mice with intact immune systems. Remarkably, while the parental 4T1 tumors were highly metastatic, metastasis from 4T1 NOS2-/- tumors was nearly eliminated. In cell culture, we find that nitric oxide (NO) from NOS2 induces a glycolytic phenotype, epitrascriptomic dysregulation, and prostaglandin E2 synthesis by stimulating COX2. Cytokine addition led to DNA damage, presumably from NO. Human colorectal cancer cell line DLD1 yielded similar results. RNA sequencing of 4T1 cells revealed a NOS2-dependent reduction in mRNAs associated with regulation of chromosome and DNA replication, including cell cycle checkpoints, as well as mRNAs associated with RNA demethylation, including tRNA demethylation. Immunoprofiling of the tumor microenvironment revealed immunosuppressed primary tumors that were low in cytotoxic T cells and high in myeloid derived suppressor cells expressing interferon gamma, which induces NOS2 expression. Taken together, these data suggest high NOS2 in tumor cells may drive metastasis through a broad accumulation of cancer-associated factors - including metabolic dysregulation, genetic dysregulation, and an increase in inflammatory prostaglandins - all of which contribute to aggressive cancers.
The integral membrane protease Rce1 cleaves prenylated CaaX proteins, including all RAS GTPases, as part of their post-translational maturation. This cleavage, which removes the -aaX residues, is necessary for the proper cellular location and signaling properties of RAS and its oncogenic forms. Poor biochemical stabilities of yeast and mammalian Rce1 have hindered the purification of Rce1 in active form and limited studies addressing its catalytic properties. Here we identify that Drosophila melanogaster Rce1 has good biochemical stability and is catalytically active when purified. The purified protein recapitulates known properties of Rce1, including specificity for prenylated CaaX substrates. Like other integral membrane proteases, the enzyme has a low turnover number (kcat), of approximately 20 hr-1. Pre-steady-state kinetic analysis reveals that product release is the rate limiting step. Our data indicate that Rce1 is inhibited by nanomolar levels of Zn2+, but that metals are not involved in catalysis. The data suggest that Zn2+ is a competitive inhibitor with respect to the substrate. The signaling lipid PA also inhibits the enzyme in vitro, raising the possibility that the cellular activity of Rce1 may be modulated by lipids and/or Zn2+. These studies combined with structural predictions and prior investigation lead to the hypothesis that the active site contains a catalytic Glu-His-His triad of amino acids that coordinate the substrate and form the inhibitory Zn2+ site. This work enhances the understanding of the enzyme and gives insight into means for Rce1 inhibition.
Lysophosphatidylcholine (LPC) is an essential mammalian phospholipid that serves multiple physiological functions. We have previously shown that plasma LPC levels are rapidly reduced after cardiac arrest (CA) in humans and in rodent models, where exogenous supplementation improves survival and neurologic outcomes. However, the specific mechanisms driving this depletion remain undefined, precluding rational design of targeted therapies to preserve LPC levels and limiting clinical translation. Here, we provide the first pharmacokinetic analysis of acute LPC reduction through a novel stable-isotope tracer approach. Our findings demonstrate that saturation status of the fatty acyl chain in LPC species determines the degree of reduction after CA, with polyunsaturated species showing the greatest declines. Further, we found that in situ metabolism, not decreased hepatic production nor increased tissue distribution, underlies the observed depletion in plasma LPC after cardiac arrest. Polyunsaturated LPC species showed correlated changes with lysophosphatidic acid and choline-containing metabolites. These correlations likely reflect shared vulnerability to post-CA pathological stress rather than direct metabolic conversion, as conventional metabolic pathways could not account for the temporal patterns and magnitude of LPC reduction, suggesting a broader, systemic dysregulation of plasma lipid metabolism. These findings indicate that therapeutic strategies must focus on either identifying and targeting the novel metabolic mechanisms responsible for species-selective elimination or implementing LPC supplementation protocols designed to overcome accelerated plasma elimination.
Small leucine-rich proteoglycans (SLRPs) are responsible for various biological functions via their multiple interactions with various proteins. Although some SLRPs form homodimers, it remains unclear how homodimerization affects the molecular functions of SLRPs. Herein, we report the molecular basis and functional significance of homodimerization of PRELP, which is a member of SLRP family. We used biophysical techniques to validate the premise that PRELP forms a reversible homodimer in a concentration-dependent manner. Because the spatial aggregation propensity (SAP) calculation indicated that the concave surface of the leucine-rich repeat domain of PRELP is highly hydrophobic, we speculated that this hydrophobicity predominantly drives PRELP homodimerization. To address this hypothesis, we replaced several amino acid residues that showed high SAP values with charged residues identified by the Rosetta energy calculation, which resulted in disruption of PRELP homodimerization. Molecular dynamics simulation further verified the molecular basis of the monomerizing mutation as well as an unexpected dimerizing mutation. To demonstrate the influence of reversible homodimerization on PRELP interactions, we conducted interaction analysis using the designed PRELP mutants. We found that the monomerizing mutation of PRELP enhanced interactions with transforming growth factor β1 and insulin-like growth factor I receptor, suggesting that the functions of PRELP that rely on these interactions are moderated via reversible homodimerization.
Oncogenic pathways do not always cooperate; in some contexts, their co-activation is antagonistic and suppresses tumorigenesis, a phenomenon we termed pathway incompatibility. However, the mechanisms underlying this antagonism and the role of receptor context in shaping these interactions remain unclear. During normal B-cell development, precursor B-cell receptor (pre-BCR) signaling supports survival and proliferation of early B-cell precursors before transition to expression of the mature B-cell receptor (BCR). B-cell acute lymphoblastic leukemia (B-ALL), the most common childhood cancer, is characterized by developmental arrest prior to BCR expression, and approximately 35% of cases harbor activating RAS-ERK mutations that mimic pre-BCR-dependent survival signaling. NF-κB plays context-dependent roles in B-cell malignancies, but whether it influences the compatibility between oncogenic RAS signaling and BCR expression remains poorly understood. Activation of canonical NF-κB induced apoptotic depletion of RAS-driven B-ALL cells. Mechanistically, NF-κB suppressed pre-BCR-dependent survival signaling while promoting expression of BCR components. Consistent with this shift, oncogenic RAS signaling was poorly tolerated in BCR-positive cells unless BCR expression was disrupted. Pharmacologic activation of NF-κB reduced ERK signaling and selectively impaired viability of RAS-driven B-ALL cells, with enhanced effects in combination with ERK inhibition. Together, these findings show that canonical NF-κB signaling promotes BCR expression, which constrains oncogenic RAS activity, and establish pathway incompatibility as a mechanism through which receptor context can limit oncogenic potential.
Neuroinflammation is a major contributor to the pathogenesis of Alzheimer's disease (AD). Although elevated serum C-reactive protein (CRP) is associated with increased AD risk, the specific conformational forms of CRP operative in the brain and their underlying mechanisms remain unclear. Here, we combined human brain transcriptomic analysis, APP/PS1 mice studies, and functional assays in BV2 cells and primary microglia to elucidate the roles of CRP and its monomeric form (mCRP) in AD-related neuroinflammation. CRP was significantly upregulated in the brains of AD patients and in the hippocampus of APP/PS1 mice, where it extensively co-localized with activated microglia. mCRP directly induced M1 pro-inflammatory microglial polarization and synergistically enhanced neuroinflammation with amyloid-β42-derived diffusible ligands (ADDLs). Additionally, mCRP dissipated mitochondrial membrane potential and promoted intracellular and mitochondrial reactive oxygen species accumulation. Mechanistically, mCRP activated the phospholipase C (PLC)-protein kinase C (PKC)-mitogen-activated protein kinase (MAPK)-nuclear factor-κB (NF-κB) signaling cascade, whereas the PLC inhibitor U-73122 attenuated pathway activation and reduced the production of pro-inflammatory cytokines. Collectively, these findings identify mCRP as a critical mediator linking systemic inflammation to microglia-driven neuroinflammation in AD, offering new mechanistic insights into AD pathogenesis.
The molybdenum cofactor (Moco) is a chemically labile prosthetic group required by a small but essential set of metazoan enzymes, including sulfite oxidase, xanthine dehydrogenase, aldehyde oxidases, and the mitochondrial amidoxime reducing components (MARC). Disruption of Moco biosynthesis in humans causes Molybdenum Cofactor Deficiency (MoCD), a severe neonatal encephalopathy. Caenorhabditis elegans is unique among animals studied so far in that it can meet its Moco requirement through both endogenous biosynthesis and direct uptake of mature Moco from its bacterial diet. However, the organism-wide abundance of the Moco biosynthetic machinery and Moco-dependent enzymes, and their response to altered Moco supply, have remained unknown. Here, using data independent acquisition proteomics with histone anchored absolute quantification, we generated an organism wide quantitative atlas of Moco biosynthesis and utilization in C. elegans under standard and Moco limiting conditions. Components of the biosynthetic pathway showed a strikingly asymmetric abundance. The mitochondrial enzyme MOC-5, which catalyzes the first committed step in Moco biosynthesis, was present at only about 120 copies per genome equivalent, roughly fifty-fold below the downstream cytoplasmic biosynthetic machinery, which ranged from about 5,000 to 8,500 copies per genome equivalent, identifying MOC-5 as a stoichiometric bottleneck. On the utilization side, the MARC paralogs were the dominant Moco consumers, with MARC-1 exceeding 20,000 copies per genome equivalent. Loss of dietary or endogenous Moco selectively depleted the nonsulfurated clients SUOX-1 and MARC-1, whereas biosynthetic proteins remained unchanged, indicating that protein stability, rather than compensatory expression, is the main response to Moco limitation.
D-alanylation of teichoic acids is a conserved modification of Gram-positive bacterial cell envelopes that modulates resistance to environmental stresses and host interactions. While the cytosolic steps of this pathway are well characterized, the extracellular reactions responsible for transferring D-alanine onto teichoic acids remain poorly understood. Here we investigate the organization of the Dlt machinery in the symbiotic bacterium Lactiplantibacillus plantarum. We determined the 2.3 Å crystal structure of the extracellular catalytic domain of DltD, which adopts an SGNH-hydrolase fold with a conserved Ser-His-Asp catalytic triad. Docking analyses with lipoteichoic acids (LTA) fragments suggest that the glycerol-phosphate backbone of LTA is accommodated along a surface groove leading to the catalytic serine, with conserved residues contributing to substrate positioning. Biochemical measurements further reveal direct interactions between DltD, the acyl-carrier protein DltX, and the LTA esterase DltE. The conserved C-terminal motif of DltX binds DltD and is required for efficient D-alanylation and for L. plantarum-mediated promotion of Drosophila juvenile growth. Together, our findings support a revised model in which DltD, DltE, and DltX form a coordinated extracellular interaction network that dynamically regulates LTA D-alanylation. This work provides new mechanistic insights into the organization of the Dlt machinery and reveals how species-specific adaptations of this conserved pathway contribute to bacterial interactions with the host.
The staphylococcal autolysin protein hydrolyzes cell wall peptidoglycans. The R2ab repeats of autolysin guide the catalytic domain by binding to cell wall components and mediate attachment to abiotic surfaces during biofilm formation on polystyrene. Characterizing protein interactions with surfaces is a significant challenge in understanding bacterial attachment. Here, using NMR spectroscopy, we determined the solution structure and backbone dynamics of the R2ab domain. We then, by combining NMR and calorimetry, identified the interaction mode of R2ab with cell wall components and polystyrene surfaces. The solution-derived R2ab structure reveals interaction surfaces with lipoteichoic acid (LTA) and peptidoglycan (PGN). The binding regions are identified, and docking is used to model their binding to R2ab. R2ab also interacts with polystyrene nanoparticles (PSNPs). Using a novel approach where PSNPs are paramagnetically labeled, we identify the major R2ab-polystyrene interaction interface. R2ab engages polystyrene surfaces via a defined C-terminal interface on R2b, as revealed by PRE mapping, ITC, and mutational analyses. Polar and charged residues (notably S776 and K779) play key roles in adsorption. These findings support a model in which R2ab acts as a molecular bridge, binding both cell wall components and abiotic surfaces. This highlights its role in early biofilm formation and suggests that disrupting this interaction could reduce S. epidermidis attachment to abiotic surfaces.
Glycoside hydrolases (GHs) exhibit remarkable specificity dictated by the structural configuration of their target glycosidic linkages. While enzymes that process α-1,4- and α-1,6-linkages in starch or glycogen are well-characterized, those acting on less common bonds, such as α-1,2-glucosidic linkages, remain largely underexplored. In this study, we report the discovery and structural elucidation of a novel α-1,2-glucosidase from Arthrobacter humicola A8F5 (A8F5 glucosidase), representing a newly uncovered activity within the poorly characterized GH176 family. Biochemical characterizations revealed that A8F5 glucosidase exclusively cleaves α-1,2-linkages via an anomer-inverting mechanism, with a preference for short kojioligosaccharides. To circumvent crystallization obstacles caused by high loop flexibility and translational non-crystallographic symmetry, we engineered a loop-truncated variant. This strategy enabled the determination of high-resolution (up to 1.79 Å) crystal structures of the enzyme in its ligand-free form and in complex with kojibiose, kojitriose, and selaginose. A8F5 glucosidase adopts an (α/α)6-barrel fold characteristic of clan GH-G. Complementing the crystal structures with AlphaFold3 prediction demonstrated that two prominent active-site loops (loops 3 and 4) adopt a closed conformation that constricts the catalytic pocket, rendering the architecture suitable for short oligosaccharide recognition while restricting access to larger polymers. Furthermore, sequence similarity network analysis highlights vast, uncharacterized functional diversity within the GH176 family. These findings revealed that the GH176 enzyme recognizes and hydrolyses α-1,2-glucosidic bonds through a structural framework distinct from that of the previously known clan GH-L GH65 kojibiose hydrolase, expanding the known functional landscape of this enzyme group toward rare α-glucans.
Gallbladder cancer (GBC) is a highly aggressive and insidious malignancy with a poor prognosis, primarily due to the lack of specific therapeutic targets and early diagnostic biomarkers. Therefore, identifying novel oncogenes involved in GBC and exploring targeted therapeutic strategies against them are of particular importance. STK31 is a cancer/testis antigen, but its biological function and mechanistic role in GBC remain unclear. This study investigates how STK31 influences GBC. STK31 expression in GBC and gastrointestinal tumors were analyzed using collected clinical samples and TCGA database respectively. Its pro-tumorigenic functions were then confirmed by in vivo and in vitro assays, while downstream regulatory mechanisms were uncovered via multi-platform proteomic strategies. Molecular docking was employed to screen 1,008 ChEMBL small molecules post STK31 functional validation. Significant upregulation of STK31 expression was observed in both GBC and gastrointestinal malignancies through our comprehensive analysis. STK31 depletion decreased GBC cell proliferation, migration and induced G1/S phase arrest and cellular polyploidization. Gene Set Enrichment Analysis (GSEA) demonstrated that STK31 functionally correlates with core biological processes including DNA Replication, Cell Cycle and Extracellular Matrix (ECM) Receptor Interaction. STK31 directly binds to both c-Myc and FOXP1, thereby regulating MCM and ITG family proteins to exert its biological function. In addition, STK31 influences the phosphorylation of c-Myc through its kinase activity. Atovaquone identified through molecular docking screening, binds STK31 and reduces its expression and kinase activity, demonstrating anti-tumor effects against GBC both in vitro and in vivo. We demonstrate that STK31 facilitates GBC progression through c-Myc/FOXP1-mediated DNA replication dysregulation. Its inhibition triggers lethal polyploidization, with ATO identified as a potential STK31-targeting drug candidate.
Glioblastoma is the most aggressive primary malignant brain tumor in adults, characterized by high invasiveness and poor prognosis. The knowledge of driver genes and prognostic markers predictive of poor outcomes in glioblastoma is still limited. In the current manuscript, we show that LAMTOR5 is a driver gene for glioblastoma progression through transcriptionally activating the HOXC9/YTHDF2 positive feedback loop. LAMTOR5 is highly expressed in glioblastoma, predicts poor prognosis in glioma patients and is essential for the growth, invasion and tumorigenesis of glioblastoma cells. Using a bioinformatics analysis approach based on multiple public datasets, we identified YTHDF2 as a downstream effector of LAMTOR5. LAMTOR5 promotes the expression of YTHDF2 through increasing its promoter activity by coactivating the transcription factor HOXC9. In turn, the LAMTOR5-upregulated YTHDF2 enhances the expression of HOXC9 through promoting the mRNA stability of HOXC9 in an m6A-dependent manner, forming a positive feedback loop in LAMTOR5-overexpressed glioblastoma cells. Silencing YTHDF2 can abrogate the LAMTOR5-promoted proliferation and invasion of glioma cells both in vitro and in vivo. Thus, we conclude that LAMTOR5 is an oncogenic protein driving glioblastoma progression via activating the HOXC9/YTHDF2 positive feedback loop. Our results offer new insights into the regulatory interplay between transcription and m6A modification of oncogenic proteins in glioblastoma and provide a therapeutic target for glioblastoma treatment.
Nitric oxide (NO) activates cGMP production by soluble guanylyl cyclase (sGC). To do so an sGCβ subunit must incorporate heme and form a heterodimer with a partner α subunit. In cells, heme-free (apo) sGCβ obtains heme from glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We recently found that transfer of ferric heme from purified GAPDH to apo-sGCβ was accompanied by heme reduction to ferrous within sGCβ, implicating redox involvement of its Cys residues. Here, we utilized Ser substitutions to probe three conserved, redox active Cys in sGCβ (C78, 122, and 214). Proteins were studied in live HEK293 cells and in purified form. In cells, C78S sGCβ was unique among the variants in being unable to incorporate either added ferric heme or mitochondrially-generated heme, but it did incorporate added ferrous heme-NO and consequently formed an active sGC heterodimer. An internal Cys oxidation event occurred in the wild type, C122S, and C214S apo-sGCβ proteins during their uptake of ferric heme from GAPDH but did not occur in C78S apo-sGCβ. We conclude: (i) Mitochondrial heme reaching apo-sGCβ in cells is ferric under normal culture conditions. (ii) C78 in the sGCβ subunit provides a reducing equivalent to the incoming ferric heme to reduce it to ferrous and this enables stable heme incorporation. (iii) No other Cys residues in sGCβ or cell thiols can replace the role of C78. Having a protein’s heme incorporation rely on an internal Cys redox event is unusual and suggests how sGC maturation may be linked to the thiol redox status in cells.
Early postnatal life is important for reproductive development, but the effects of early-life nutrition on female reproductive capacity remain largely unknown. Here, we investigated whether neonatal ketone body deficiency affects adult oocyte quality using a lactational malnutrition model and Hmgcs2-deficient mice. We found that impaired neonatal ketogenesis caused long-lasting impairments in female reproductive function, including reduced ovarian reserve, decreased oocyte developmental competence, altered hormone levels, and impaired embryo development. Neonatal β-hydroxybutyrate (β-HB) supplementation partially improved these reproductive abnormalities, indicating an important role of ketone metabolism during early ovarian development. Transcriptomic and epigenomic analyses showed that Hmgcs2 deficiency disrupted histone H3 acetylation homeostasis in oocytes. These Ace-H3 changes were enriched in genes related to oxidative stress and apoptosis and were accompanied by transcriptional alterations. β-HB supplementation partially restored the altered Ace-H3 patterns. Together, our findings reveal that ketone bodies regulate oocyte development by connecting early-life nutrition with epigenetic regulation.
Siglec-1 (Sialoadhesin/CD169), expressed on myeloid cells, is a receptor for sialic acids that mediates adhesion to host cells and pathogens. However, certain pathogens exploit Siglec-1 binding for further dissemination and interfering with this binding may serve as potential therapeutic interventions. Here we identified high-affinity single domain antibodies, (also known as VHHs or Nanobodies) that allo- or orthosterically modulate ligand binding of Siglec-1. VHH clone 2C2 bound directly to the ligand binding site of Siglec-1 and effectively blocked binding of a diverse set of ligands, such as ganglioside liposomes, Campylobacter jejuni, and SARS-CoV-2, to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1+ DCs. In contrast, VHH 1B5 and 1C1 bound outside of the ligand binding site and enhanced Siglec-1 interactions with ganglioside liposomes and Campylobacter jejuni. Mechanistically, VHH 1B5 and 1C1 appear to interfere with the cis-binding sialic acids present on the Siglec-1-expressing cell surface, thereby facilitating increased trans-interactions with ligands. In conclusion, we have isolated VHH that bind to different sites on Siglec-1 and thereby enhance or block ligand binding to a variety of sialylated pathogens. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH may serve as broad-spectrum pathogen blocking agents.
Lactylation, a novel post-translational modification involving lactate conjugation to lysine residues, bridges metabolic flux and epigenetic regulation and has emerged as a conserved regulatory mechanism across eukaryotes and prokaryotes, governing cell proliferation, immune responses, and metabolic reprogramming via a “Writer-Eraser” system. Despite the established roles of lactylation in energy metabolism, its function in male reproduction remains unexplored. This study investigates the lactylation modification dynamics during human sperm capacitation, a process requiring metabolic reprogramming for hyperactivated motility. Through 4D FastDIA-based quantitative dynamic profiling, we firstly conducted the comprehensive characterization of lysine lactylation modifications during sperm capacitation, systematically mapping differential lactylation patterns at the pathway, proteomic, and site-specific levels. We identified 2,030 quantifiable lactylated proteins, revealing significant capacitation-dependent modulation: 115 proteins with 133 sites demonstrated upregulated lactylation, while 287 proteins containing 471 sites showed downregulation (P < 0.05, Fold Change > 1.5). Notably, 30 proteins exhibited bidirectional regulatory patterns with coexisting upregulated and downregulated sites, and a predominant proportion of modified proteins (263 of 372) displayed single-site differential lactylation. A marked reduction in lactylation levels of mitochondrial respiratory chain proteins was observed after sperm capacitation, indicating heightened ATP requirements. Moreover, proteins with differential lactylation were enriched in biological processes including fertilization, sperm capacitation, β-oxidation, and microtubule dynamics. Strikingly, the presence of known lactylation regulatory enzymes including Alanine--tRNA ligase 1 (AARS1), Sirtuin (SIRT) 2/3, and Histone deacetylase (HDAC) 1/2/6 were detected in the sperm proteome, revealing a 1.6-fold upregulation of SIRT3, implicating its role in delactylation regulation during capacitation. Collectively, these findings delineate a lactylation-coordinated mechanism governing mitochondrial oxidative phosphorylation optimization and cytoskeletal architecture remodeling. This work substantiates a trinity regulatory mechanism integrating "metabolism-epigenetics-function" axes through lactylation circuitry, which may have implications for understanding male infertility.
The force-generating ATPases Associated with diverse cellular Activities (AAA+) ATPase domain of protein unfoldases is specified for many substrates and other functional partners through elaboration with accessory domains. Mitochondrial homologs of the unfoldase ClpX contain an insertion within the AAA+ domain that is absent in bacterial homologs. This mitochondrial insertion (MI) maps to the substrate-encountering face of ClpX, leading us to hypothesize that the MI directs interactions of ClpX with mitochondrial substrates, the best-characterized of which is the first enzyme in heme biosynthesis, 5-aminolevulenic acid synthase (ALAS). We find that the MI is critical for both recruitment and activation of ALAS by S. cerevisiae ClpX. The MI was dispensable for heme-induced, adaptor-mediated degradation of ALAS by the human CLPX-CLPP protease complex (CLPXP), but contributed to adaptor-independent recruitment of the model substrate casein for degradation. Although truncation of the MI moderately perturbed ATPase activity in both yeast and human ClpX, this effect could be uncoupled from the requirement for the MI in the efficiency of ALAS activation by targeted mutagenesis. The MI therefore can serve both to recruit a substrate to mitochondrial ClpX and to accelerate its processing by the AAA+ motor.
