Journal of Biological Chemistry最新文献

Cell-scale actin remodeling requires rapid actin depolymerization beyond that generated by cofilin and gelsolin. Previous reports had indicated that the activity of myosin restricted the length of actin bundles. However, it was unknown whether the ubiquitous non-muscle myosin II isoforms (NMIIA and IIB) could generate cell-scale actin dynamics. Using linear actomyosin network simulation, we observed higher network tension and faster network rupture with NMIIA when compared to NMIIB. Live cell imaging of the actin network in COS7 cells also showed a similar result with numerous network severing events recorded in the presence of NMIIA while NMIIB produced fewer bundle severing events. Moreover, NMIIA was required for the formation of peripheral actin arcs and long actin fibers that were absent in cells expressing NMIIB. We also observed the peripheral localization of cofilin in the presence of NMIIA supporting the live cell imaging data of increased actin severing by NMIIA. Finally, using fluorescence recovery after photobleaching (FRAP), optical trap based cortical force measurements and live cell imaging of actin network, we showed that the dynamics of the actin network increased with the increasing expression of NMIIA but not NMIIB. Thus, we established NMIIA as the predominant generator of cell scale actin dynamics.

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, occurs in all three domains of life and plays key roles in metabolism and regulation. While polyP metabolism has been well studied in bacteria and eukaryotes, studies in archaea have been limited, where the polyphosphate kinases (PPKs) involved in polyP synthesis remained largely uncharacterized. Notably, members of the Crenarchaeota (Thermoproteota) lack homologs of bacterial PPKs. We identify two genes in the crenarchaeal model organism Sulfolobus acidocaldarius, (saci_2019 and saci_2020), previously annotated as thymidylate kinases, that together encode a heteromeric archaeal PPK (SaPPK3). Saci_2019 acts as the catalytic subunit (cPPK3), whereas Saci_2020 is a regulatory subunit (rPPK3) that enhances activity through oligomerization. SaPPK3 is reversible but strongly favors polyP-dependent nucleotide kinase activity, forming ATP from ADP and polyP. Kinetic modelling combined with quantitative ³¹P NMR showed that polyP synthesis occurred only at high ATP/ADP ratios in the presence of an ATP recycling system, suggesting that SaPPK3 promotes ATP production from polyP under low energy conditions in vivo. Enzymatic, structural and phylogenetic analyses place SaPPK3 in a distinct PPK family within the thymidylate kinase superfamily of P-loop kinases. The PPK3 family members show a patchy distribution, being represented mainly in Crenarchaeota of the families Nitrososphaeraceae and Sulfolobaceae, and in a few bacteria. Our findings identify PPK3 as a critical missing link enzyme involved in archaeal polyP metabolism and suggests that polyP, in addition to its function in phosphate storage, serves as an emergency energy buffer.

Human leukocyte immunoglobulin-like receptors (LILRs) are cell surface receptors that are mainly expressed in immune cells. LILRs are involved in immune cell regulation. As a member of the LILR family, LILRA2 was reported to recognize the bacterially N-terminus truncated Ig (N-truncated Ig) for the induction of innate immune response. Fibrinogen, which is enzymatically converted to fibrin and forms fibrin-based blood clots, was recently shown to activate LILRA2-expressing immune cells. However, the molecular mechanisms of LILRA2-fibrinogen interaction remain unclear. In this study, we investigated the molecular recognition of fibrinogen by LILRA2, using biophysical methods. Surface plasmon resonance (SPR) analysis showed that LILRA2 specifically bound to fibrinogen with a relatively low dissociation constant (K_D) (∼ 10 μM) like N-truncated Ig. Furthermore, we found that high-molecular-weight fibrinogen exhibited a high-affinity interaction with immobilized LILRA2 owing to significant avidity effects. Domain-deletion and site-specific mutagenesis successfully identified the crucial amino acids of domains 2 and 4 of LILRA2 for fibrinogen binding. On the other hand, D regions of fibrinogen is responsible for binding to LILRA2. These results enabled us to build a reasonable model of the LILRA2-fibrinogen complex, which provides insights into the molecular recognition and therapeutic potential of LILR-mediated immune responses.

The plasma membrane (PM) is a dynamic interface that integrates environmental cues with cellular responses. Insulin is known to remodel the PM primarily by stimulating the translocation of glucose transporter GLUT4, but the full scope of insulin's PM remodeling remains poorly defined. Here, we performed a meta-analysis of insulin-regulated PM proteins in adipocytes by integrating nine independent proteomic datasets generated using complementary PM enrichment strategies. The meta-analysis identified 37 insulin-regulated candidates detected in at least three datasets, including 30 proteins not previously implicated in insulin action. Among these, we experimentally characterized the insulin-stimulated translocation of two transporters: potassium-chloride cotransporter 1 KCC1 (SLC12A4) and sodium-dependent phosphate transporter PIT2 (SLC20A2), which showed robust and reproducible recruitment to the PM in response to insulin. siRNA-mediated knockdown of KCC1 or PIT2 impaired insulin-stimulated glucose transport, suggesting a role for these transporters in insulin action. Live-cell and fixed-cell imaging revealed that both proteins localize across multiple endosomal compartments, undergo insulin dose-dependent trafficking to the PM, and require PI3K-AKT signaling for their mobilization. Strikingly, insulin-induced translocation of KCC1 and PIT2 to the PM was impaired in adipocytes rendered insulin resistant by chronic hyperinsulinemia, accompanied by increased perinuclear retention under basal conditions. Together, our work provides a valuable resource for understanding insulin-regulated PM remodeling in adipocytes, establishes KCC1 and PIT2 as novel insulin-responsive transporters, and supports the idea that insulin resistance involves defects in cell-surface delivery that extend beyond GLUT4.

Diverse bacteria possess unusual gene clusters containing cryptic genes of unknown function, which are often related to metabolism of sugars and sugar acids. In 1964, Aspen and Jakoby first isolated and characterized an NAD⁺-dependent L-threonate 3-dehydrogenase (Ltn3D; EC 1.1.1.29) from Pseudomonas sp. (J Biol Chem 239, 710-713), the molecular identity of which has remained unknown for over 60 years. Here, we have utilized bacterial genome context, together with biochemical and structural characterization, to reveal that GL300_RS07945 in Paracoccus litorisediminis encodes a representative NADP⁺-preferring Ltn3D. Crystal structure of the Michaelis ternary complex indicated that this enzyme is a member of the short-chain dehydrogenases/reductase superfamily, yet differed in the recognizing of the 2'-phosphate group of NADP⁺ between two adjacent arginine residues (Arg33 and Arg34). The C-3 atom of the competitive inhibitor tartronate was rationally positioned in close proximity to the nicotinamide ring for the catalysis. The reaction catalyzed by Ltn3D constitutes a distinct bypass route for the direct conversion of L-threonate to 3-oxo-L-threonate, which differs from the known sequential steps involving a dehydrogenase (Ltn2D) and an isomerase (OtnI). In contrast to Ltn2D, Ltn3D efficiently oxidized the 3-OH of homologous five- and six-carbon sugar acids, in addition to L-threonate. Among them, D-gluconate, potentially produced by GL300_RS07940 as a bifunctional 2-keto-D-gluconate/2-keto-L-gluconate reductase, could be converted to D-ribulose 5-phosphate by Ltn3D followed by the action of a kinase (3OtnK) and a decarboxylase (3OtnC) in vitro. Altogether, our data suggest that Ltn3D constitutes a unique evolutionary innovation for the catabolism of four- to six-carbon sugar acids.

Pumilio proteins are conserved RNA-binding proteins that control mRNAs involved in development, proliferation, and differentiation. Human PUM1 and PUM2 repress targets by recruiting the CCR4-NOT deadenylase complex through a metazoan-specific, intrinsically disordered repression domain (RD3). Here we dissect RD3 using functional assays, protein interaction assays, and crosslinking mass spectrometry. We identify multiple RD3 peptides that are sufficient for repression and binding to the CCR4-NOT complex. Crosslinking reveals numerous mutually exclusive contacts between RD3 and CCR4-NOT, consistent with a multivalent "fuzzy" binding mode in which interactions are not defined by a single sequence or structure. Sequence scrambling shows that the linear amino acid order of RD3 is dispensable, whereas its physicochemical composition, in particular aliphatic and aromatic residues, is essential for repression and CCR4-NOT binding. These findings support a model in which multivalent interactions between intrinsically disordered regions (IDRs) and effector complexes, governed by amino acid composition, underlie robust PUM-mediated repression, and exemplify general principles by which IDRs recruit CCR4-NOT to regulate gene expression.

The incidence of early-onset gastric cancer (EOGC) is increasing. While RNA alternative splicing (AS) critically regulates cancer progression, and abnormal changes in splicing factors (SFs) can affect AS regulation, their roles in EOGC remain unclear. Using multi-omics approaches, we explored the expression and regulatory patterns of 75 SFs in EOGC and further analyzed the differences associated with different regulatory patterns. We investigated the role of serine/arginine-rich splicing factor 1 (SRSF1) in regulating oxaliplatin (OXA) resistance and malignant phenotypes in EOGC. The results showed that the expression levels of most SFs in the EOGC samples were significantly upregulated, while the somatic mutation rate of SFs was low. Based on the expression of SFs, the EOGC population can be stably divided into three splicing regulatory patterns, which differ in immune function, tumor mutational burden, and the anticipated response to chemotherapy drugs. Overexpressing SRSF1 confers OXA resistance to EOGC cells, promotes colony formation, and inhibits apoptosis, and it could promote exon skipping in downstream genes, thereby altering tumor-related functions. This study reveals the expression landscape of SFs in EOGC and highlights the disparities in biological functions across various splicing regulatory patterns. SRSF1 could be a potential therapeutic target and biomarker for overcoming OXA resistance in EOGC.

Copper (Cu) is an essential trace element required for mitochondrial respiration via its incorporation into cytochrome c oxidase (CuCOX), the terminal enzyme of the electron transport chain. Here, we employed size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS), UV-Vis spectroscopy, and immunoblotting to identify and validate a high molecular weight Cu-containing peak in the SEC-ICP-MS chromatogram as representative of CuCOX activity. We demonstrate that this CuCOX peak is enhanced under metabolic conditions inducing oxidative phosphorylation, such as high Cu supplementation or galactose-containing media, and correlates with increased mitochondrial respiration. Using exogenous ⁶³Cu tracing, we characterized the time- and dose-dependent incorporation of newly acquired Cu into CuCOX under elevated Cu conditions in renal cancer cells, modeling advanced clear cell renal cell carcinoma (ccRCC). RNA interference experiments targeting key Cu transporters revealed that CuCOX formation is independent of the high-affinity Cu importer CTR1, but instead relies on alternative transporters, including DMT1, LAT1, and the mitochondrial carrier SLC25A3, with transporter contributions dynamically reshaped during chronic adaptation to high Cu availability. In contrast, under standard low-Cu conditions, CTR1 remains required for cellular Cu uptake and CuCOX metallation. Together, these findings define context-dependent Cu trafficking pathways in renal cancer and establish SEC-ICP-MS as a sensitive platform for assessing CuCOX metallation and mitochondrial metabolism, with potential applications in biomarker discovery and therapeutic targeting in RCC.

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.