Journal of Biological Chemistry最新文献

Limb-girdle muscular dystrophy R1 (LGMDR1) is an autosomal recessive disorder caused by dysfunction of calpain-3 (CAPN3; also known as p94), a muscle-specific, Ca²⁺-dependent cysteine protease. LGMDR1 mutations are distributed throughout the Capn3 gene. Nevertheless, our knowledge of the biochemical and biological properties of individual LGMDR1 mutants is limited, hindering a full understanding of LGMDR1 pathogenesis. Here, we comprehensively examined the functional properties of LGMDR1 mutants within the penta-EF-hand (PEF) domain at the COOH-terminus of CAPN3, focusing on their autolytic processing, oligomerization, titin binding, and subcellular localization within sarcomeres of mouse skeletal muscle. We found that oligomer formation of CAPN3 through the PEF domain contributes to efficient NH₂-terminal and IS1-region processing, which were impaired by specific LGMDR1 mutations within the PEF domain. Furthermore, while wild-type CAPN3 predominantly localized at the sarcomeric M-bands of tibialis anterior muscles in vivo, several LGMDR1 mutants were absent from the M-bands due to decreased binding to titin, a giant cytoskeletal protein, irrespective of their oligomerization status. These findings indicate that LGMDR1 mutations within the PEF domain disrupt the physiological function of CAPN3 through both oligomer-dependent and -independent mechanisms, highlighting two distinct pathways contributing to LGMDR1 pathogenesis.

The pyruvate dehydrogenase complex (PDHc) is a universally conserved multienzyme system that converts pyruvate into acetyl-CoA for entry into the TCA cycle and for NADH production. Its central scaffold, the dihydrolipoyl transacetylase (E2p), forms an oligomeric inner core that recruits pyruvate dehydrogenase (E1p) and dihydrolipoyl dehydrogenase (E3). All previously characterized PDHc assemblies adopt either an octahedral 24-mer or an icosahedral 60-mer E2p core, each constructed from trimeric building blocks. We recently showed that the Mycobacterium tuberculosis (Mtb) E2p protein DlaT also functions as the core of the pathogen's peroxynitrite reductase/peroxidase (PNR/P) complex. Here, using cryo-EM, we demonstrate that DlaT assembles into discrete hexamers and dodecamers at micromolar concentrations, which approximate intracellular DlaT concentrations in Mtb. Structure-guided mutagenesis combined with in vitro activity assays indicate that the hexamer represents the functional E2p core of the Mtb PDHc. This noncanonical architecture arises from unique interfaces between DlaT trimers that preclude formation of the classic spherical 24- or 60-mer structures. We propose that this specialized E2p organization enables Mtb to regulate metabolic activities and to remodel the E2p core for engagement in the PNR/P antioxidant pathway under stress. Our findings reveal an unexpected diversity in PDHc architecture and uncover a distinct organization principle for the core metabolic complex in mycobacteria.

Lamins are critical in maintaining nuclear homeostasis, chromosome positioning, and modulating mechanotransduction. Recent studies indicated the involvement of lamin A in mitochondrial homeostasis and the regulation of superoxide. Missense mutations in LMNA are linked to a spectrum of diseases known as laminopathies, which include conditions such as Dilated Cardiomyopathy (DCM), muscular dystrophy, and progeria. K97E is one such mutation, which leads to DCM with severe phenotypes. In this study, we established direct reduction of interaction between lamin A K97E and PHB2. As a sequel, mitochondria exhibited reduced fusion, elevated fragmentation and ATP deficiency. On the other hand, impaired RhoA-ERK-FAK signalling cascade disrupted F-actin assembly thereby promoting actin-mitochondria association, further facilitating mitochondrial fission. This feedback-loop led to mitochondrial depolarisation, and global metabolic derangement, in particular, reduced glycolytic capacity, incomplete fatty acid oxidation accompanied by elevated superoxide levels. In cardiomyocytes, such dysfunction may be correlated with contractile defects and arrhythmias. Thus, our findings elucidated for the first time the pivotal role of lamin A in cellular-bioenergetics and mechanotransduction, offering novel insights into DCM pathophysiology, which could open newer vistas for developing targeted therapeutic strategies.

Processing bodies (PBs) are cytoplasmic granules that function in the cellular response to stress conditions by regulating mRNA metabolism. Initially, they were thought to represent sites of mRNA turnover, whereas more recent work points to a role in the storage of useful mRNAs. However, their exact intracellular role remains unclear. We used SH-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) to study PB-localisation and global mRNA fate during glucose depletion conditions that induce PB formation in yeast. This enabled us to differentiate newly synthesized and pre-existing RNAs and to separately track mRNA synthesis and degradation. We show that pre-existing mRNAs localise to PBs with differing kinetics with some transcripts localising over the time-course of glucose starvation and some transcripts localising in a more dynamic manner. We identified a small number of transcripts that are enriched only transiently in PBs, consistent with the traditional view of PBs acting as sites for RNA decay. However, most transiently localised transcripts are not destabilized following glucose starvation, with PBs appearing to act as temporary storage sites for transcripts that later undergo alternative fates. For other transcripts, both their pre-existing and newly made transcripts accumulate in PBs over the time-course of glucose depletion and we suggest that these transcripts are important for adaptation once the nutrient stress is relieved. Together, our data indicate a model where transcripts partition into different classes that behave differently following nutrient depletion with PBs acting as triage sites for mRNAs to direct their fate.

Prostaglandin H₂ (PGH₂) is formed from arachidonic acid by cyclooxygenases (COX) and metabolized by thromboxane (TXS) and prostacyclin synthases (PGIS), two self-sufficient cytochromes P450 (CYP). The related fungal linoleic acid (LA) biosynthetic route is catalyzed by di-heme proteins of five dioxygenases (DOX) fused to three akin CYP subfamilies of allene oxide (AOS), linoleate diol (LDS), and epoxy alcohol (EAS) synthases. AlphaFold2 predicted the 3D structures of the DOX-CYP domains with very high confidence. Superposition with the COX:LA enzyme complex indicated that the protein fold of central α-helices and the motifs of the substrate recognition sites (SRS) were conserved, which suggest evolution from an ancient peroxidase precursor. TXS, PGIS, and AOS catalyze homolytic scissions of oxygen-oxygen bonds and LDS/EAS heterolytic scissions. The SRS4 of LDS and EAS predicted an Asn residue at close distal axial position of the heme thiolate iron in analogy with PGIS and plant AOS, but a nonpolar in TXS and 8S/9S-AOS, and a polar (Thr) in 8R/9R-AOS. Replacements of amide residues in SRS4 of LDS shifted the position of intramolecular hydroxylation of 8R-hydroperoxy-LA and the heterolytic scission to towards homolytic. The self-sufficient CYP may catalyze homo- and heterolytic cleavage of hydroperoxides and the endoperoxide of PGH₂ by different mechanisms, but the presentation of the oxygen-oxygen bonds to the metal centers might be crucial. The AF2 models illustrate the structural, catalytical, and evolutionary relationships between COX and microbiological DOX with CYP companions in unprecedented details, which reveal multiple amino acids of potential catalytic significance for future research.

Mitochondrial AAA+ proteases, LONP1, ClpXP, YME1L (i-AAA), and the m-AAA complex, maintain protein quality and shape organelle function. Growing interest in these enzymes stems from their association with neurodegeneration, cardiomyopathy, metabolic disease, and cancer. Recent structural and biophysical work clarifies how ATP-driven conformational cycles enable substrate recognition, unfolding, translocation, and proteolysis, and how assembly state, subunit composition, and regulatory inputs tune activity. These insights help interpret patient variants and guide experiments that connect mechanism to phenotype. Here we review shared mechanistic principles across the four proteases, contrast their architectures and regulatory features, and relate these properties to substrate selection and disease mechanisms, with emphasis on evidence from structural, biochemical, and cellular studies. We also survey strategies to modulate function. Small molecules, exemplified by Dordaviprone (ONC201) which activate human ClpP, provide proof of concept, and emerging modalities such as engineered macromolecules, may offer the selectivity and localization required to correct disease mechanisms or exploit disease dependencies. By integrating mechanism, disease links, and modulation strategies, this review provides a framework for translating basic insight on mitochondrial AAA+ proteases into new tools and, ultimately, therapies.

Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation in congenital CMV infection. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. We further observed that CMV-encoded IE1 protein interferes with host PML-DISC1 interaction, resulting in disturbance of the Notch pathway in vitro and in embryonic brains. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.

Sialic acid (Sia) is essential for human physiology and health, as emphasized by the range of human diseases that is linked to abnormalities in the Sia pathway. Sias are typically found at the outermost part of glycoconjugates that are involved in several biological processes, including cell adhesion and signaling. Sia metabolism is key to the production of CMP-Sia, the building block for sialylation, and is targeted as a therapeutic strategy to ameliorate the effects of abnormal sialylation in disease. Interestingly, patients with different genetic defects in Sia metabolism show contrasting clinical symptoms affecting different tissues. For example, neurological symptoms are dominant in some congenital disorders of glycosylation (CDGs) like NANS-CDG, while the brain is unaffected in GNE myopathy which presents with isolated muscle symptoms. This suggests that more complex tissue-specific regulatory mechanisms may exist. In this review, we discuss the biosynthetic and genetic pathways in Sia metabolism with a specific focus on its role in brain, muscle, and platelets in health and genetic disease. Moreover, this review presents an overview of the clinical symptoms and genetic spectrum for each genetic disease. Overall, the molecular an biochemical profiles are not fully understood in these patients and effective therapies are limited. Therefore, additional research should focus on unravelling metabolic mechanisms that could be targeted to develop novel therapeutic strategies.

Neuropathic pain is a chronic condition characterized by damage to and dysfunction of the peripheral or central nervous system. There are currently no effective treatment options available for neuropathic pain, and existing drugs often provide only temporary relief with potential side effects. Multilineage-differentiating stress-enduring (Muse) cells are characterized by high expansion potential, a stable phenotype and strong immunosuppression. These properties make them attractive candidates for therapeutics for neuropathic pain management. Muse cells from different species demonstrated analgesic potential by reversing chronic constriction injury model (CCI)-induced neuropathic pain. Protein profiling revealed a high degree of similarity between Muse cells and bone marrow stromal cells (BMSCs). The intrathecal injection of Muse cells effectively reduced neuropathic pain in various mouse models, resulting in better analgesic effects than the administration of equivalent low doses of BMSCs. Immunohistochemical analysis and qPCR revealed the ability of Muse cells to inhibit spinal cord neuroinflammation caused by spared nerve injury model (SNI). In addition, Transwell and ELISA revealed that Muse cells migrated through the injured dorsal root ganglion (DRG) via the CCR7-CCL21 chemotactic axis. In addition, the secretion of TGF-β and IL-10 by Muse cells was identified as the mechanism underlying the analgesic effect of Muse cells. The capacity of Muse cells to mitigate neuroinflammation and produce analgesic effects via the modulation of TGF-β and IL-10 underscores their potential as promising therapeutic approaches for the treatment of neuropathic pain.