Journal of Biological Chemistry最新文献

Ferroptosis is a non-apoptotic cell death characterized by iron-dependent lipid peroxidation and is implicated in renal diseases, including acute kidney injury and diabetic nephropathy. In renal proximal tubular cells, the regulation of ferroptosis is particularly critical for maintaining cellular homeostasis. While inducing ferroptosis in normal rat kidney proximal tubular epithelial (NRK-52E) cells, we observed the emergence of resistant subpopulations and established two ferroptosis-resistant clones, designated clone A and clone B, to investigate the underlying mechanisms. Transcript and immunoblot analyses revealed that clone A lacked acyl-CoA synthetase long-chain family member 4 (ACSL4), and this deficiency conferred ferroptosis resistance. Furthermore, although clone B expressed ACSL4, its enzymatic activity was markedly reduced, leading us to hypothesize that clone B harbors a mutation that impairs ACSL4 function. Therefore, we performed sequence analysis and identified a novel T237A missense mutation in ACSL4. Sequence alignment and structural superposition of rat ACSL4 and long-chain fatty acyl-CoA synthetase from Thermus thermophilus suggested that Thr237 in rat ACSL4 cooperates with Glu429 in Mg²⁺ coordination. Functional assays using ACSL4-deficient cells expressing ACSL4 variants (T237A or E429A) confirmed that both residues are essential for catalytic activity. These findings provide new insights into the structural and functional roles of mammalian ACSL4 and may facilitate the development of ACSL4-targeted therapeutics.

Hepatic fatty acid oxidation (FAO) is crucial for maintaining hepatic lipid homeostasis, and dysregulation of hepatic lipid metabolism is closely associated with metabolic dysfunction-associated fatty liver disease (MASLD). However, the molecular mechanisms governing FAO in hepatocytes remain incompletely understood. Here, we demonstrate that RNF90 is essential for FAO regulation, with its expression significantly upregulated in fatty liver. Further investigation revealed that RNF90 is transcriptionally activated by KLF5 and functions as an E3 ubiquitin ligase to promote the ubiquitin-dependent proteasomal degradation of CPT1α, a rate-limiting enzyme in mitochondrial FAO. Hepatocyte-specific RNF90 knockout significantly increased CPT1α protein expression, enhanced FAO activity, and alleviated hepatic steatosis, as evidenced by reduced hepatic lipid accumulation and TG levels. Conversely, overexpression of wildtype RNF90 (but not its E3 ligase-deficient mutant) exerted the opposite effects. Function rescue experiments further confirm that CPT1α is indispensable for RNF90-mediated regulation of FAO and protection against hepatic steatosis. Collectively, our study establishes RNF90 as a critical regulator of hepatic lipid metabolism and identifies the KLF5/RNF90/CPT1α axis as a potential therapeutic target for MASLD.

KRAS4a and KRAS4b are important regulators of signaling, and their interactions with the plasma membrane are dynamic and influenced by lipid composition. KRAS 4a and 4b have nearly identical globular domains but differ in their membrane associated hyper variable region (HVR). The functional distinctions between these isoforms remain unclear, particularly with regards to their dependence on specific lipids and the membrane environment. Previous work showed that the membrane orientation of KRAS4b affects its ability to bind to RAF kinase RBDCRD and that the KRAS-RBDCRD complex adopts different poses on the membrane as well as influences the size and composition of the lipid environment. To model differences between KRAS 4a and 4b protein-lipid interactions, we extended the Multiscale Machine-Learned Modeling Infrastructure (MuMMI) to incorporate continuum simulations in the grand canonical ensemble, enabling sampling across macroscopic, coarse-grained, and all-atom resolutions. Using this framework, we systematically altered PIP2 concentrations, KRAS 4a vs. 4b, and RAF RBDCRD complexation to assess impacts on membrane-protein interactions and dynamics. Our results reveal that reducing PIP2 shifts and broadens the membrane orientational preference of both KRAS 4b and 4a, with stronger effects on 4b HVR localization versus 4a. We demonstrate that with depletion of the strong negatively charged PIP2 lipid, the less charged phosphatidylserine (PS) replaces PIP2. Our findings highlight similarities and distinctions in the dynamics and lipid dependency of KRAS isoforms and suggest that ordering of the local lipid composition by HVRs is a shared property and key modulator of RAS-mediated signaling at the plasma membrane.

The Target of Rapamycin (TOR) kinase is a core component of two evolutionarily conserved complexes, TORC1 and TORC2, which regulate growth, metabolism, and stress responses. In Schizosaccharomyces pombe, TORC2 is dispensable for proliferation under optimal conditions but is essential for survival and adaptation to a variety of stress conditions, including DNA damage and replication stress. The MluI-binding factor (MBF) transcription complex regulates G1/S progression and the DNA replication stress response. Previously, we demonstrated that TORC2-Gad8 is required for the upregulation of MBF-dependent gene transcription in response to replication stress. Here, we show that in response to replication stress TORC2 is necessary for the accumulation of the initiating form of RNA polymerase II (Pol II) at MBF promoters. In contrast, the elongating form of Pol II aberrantly accumulates at MBF coding regions in TORC2-deficient cells under both induced and non-induced conditions, suggesting a defect in balancing Pol II initiation and elongation that leads to impaired MBF gene induction. Unexpectedly, TORC2-deficient cells also exhibit aberrant upregulation of stress-activated genes during replication stress, including a distinct subset of Pap1-dependent oxidative stress genes. Consistent with this, TORC2 mutant cells accumulate reactive oxygen species in response to replication stress. Together, our findings suggest that TORC2 is required to ensure proper upregulation of MBF-dependent gene transcription during replication stress, and to suppress inappropriate activation of oxidative stress response pathways.

The fidelity of assembly of multiprotein complexes is essential for the formation of stable and functional protein complexes that are critical for cell growth and survival. In this context, TBP-associated factor (TAF) subunits maintain tight specificity for their integration into TFIID and SAGA complexes. In this work, using affinity purification-coupled mass spectrometry of epitope-tagged TFIID subunits TBP and TAF11, and the SAGA subunit TAF12L we identified components of the C. albicans TFIID and SAGA complexes. Whereas TAF12 is a subunit of TFIID, the paralogous TAF12L is a subunit of the SAGA complex, and we further identified each of the TFIID and SAGA complex subunits with high confidence. We found that the steady-state levels of the histone fold domain containing pairs, TAF12-TAF4 and TAF12L-Ada1 proteins, are mutually dependent on the stable expression of each other. Using RNA immunoprecipitation from polysome-containing extracts, we found that nascent TAF4 and Ada1 proteins interact with TAF12 and TAF12L, respectively, by a cotranslational mechanism in an ordered, sequential mode of assembly. Our results further revealed that the intrinsic position of the histone fold domain within the protein sequence is crucial for determining the sequence and directionality of cotranslational assembly, ensuring both selectivity and stability of the histone fold domain containing heterodimeric proteins in the fungal pathogen C. albicans.

Chordates have adapted to diverse thermal environments, with poikilotherms adjusting to external temperatures and homeotherms maintaining stable body temperatures. While housekeeping enzymes conserve their activities, they function at different body temperatures among species. However, the determinants for the optimum temperatures of housekeeping enzymes largely remain unclear. In this study, we identified the determinants of the optimum temperatures of chordate adenylate kinase 1 (AK1), a key housekeeping enzyme. The optimum temperatures of AK1s were shown to be closely correlated with the normal body temperature of each chordate (11 species). A combination of enzymatic assays, computational analyses of numerous physicochemical interactions, and structural dynamics analyses of intact and mutant AK1s verified that the number of hydrophobic interactions among four amino acids in specific secondary structures (the 4th-17th regions) is a major determinant of the optimum temperatures of chordate AK1s. This allowed us to generate a predictive model for the optimum temperatures of native chordate AK1s: AK1 optimum temperature = 2.3587× (the number of 4th-17th interactions) + 0.3663× (the number of 13th-17th interactions)- 5.9695, with a maximum error of ±1.70°C. In contrast, sequence similarity and molecular phylogenetic relationships did not correlate with the optimum temperatures of chordate AK1s. Furthermore, these results suggest an evolutionary scenario for thermal adaptation of chordate AK1. Collectively, our study provides evidence that subtle hydrophobic interactions play a crucial role in determining the temperature preferences of a chordate housekeeping enzyme, offering new insights into the functional evolution and diversification of enzyme homologs.

Skeletal muscle atrophy occurs in diverse conditions, including aging, disuse, cancer cachexia, and chronic disease. It results from an imbalance between protein synthesis and degradation, where excessive proteolysis drives loss of contractile proteins, weakness, and metabolic decline. Recent advances in structural biology, multi-omics approaches, and high-resolution imaging have uncovered how sarcomeric and cytoskeletal components are gradually degraded by ubiquitin ligases, proteasomes, and autophagy. Mechanical loading and mechanotransduction emerge as key regulators of proteostasis, linking tension to anabolic signaling. Transcriptional and epigenetic control through IGF1-Akt-mTOR, TGF-β, inflammatory cytokines, and circadian rhythms as well as non-coding RNAs and miRNAs also contribute to wasting. This review summarizes these recent findings, and novel therapeutic strategies such as restoring mitochondrial function and modulating RNA networks and mechanosensitive signaling to preserve muscle mass and function.

Tauopathy, including Alzheimer's Disease, is the most prevalent neurodegenerative disorder, yet current therapies fail to halt disease progression, partly due to limited understanding of early Tau pathological structures. Cryo-electron microscopy has clarified distinct Tau structures tied to disease phenotypes, but only in their mature forms and at low throughput. Hence, molecular probes that can recognize early Tau conformations in high throughput and in situ hold the potential to transform our understanding of Tau aggregation. Tau undergoes sequential changes, including hyperphosphorylation and misfolding, with the aggregation-prone PHF6 region in R3 shown to drive its self-assembly and fibrillar core formation. In this study, we used existing and novel monoclonal antibodies (mAbs) to map the Tau protein in AD and other Tauopathies. The conformation-specific antibody 16B12, targeting the R1-R3 regions, showed high sensitivity in detecting early Tau structures and inhibited seed-induced aggregation in vitro. Another antibody, 9H6F2, detected P1-P2 interactions and functioned as a pan-biomarker across Tauopathies. Our findings underscore the critical role of early P1-P2 and R1-R3 interactions in Tau fibril maturation, and our mAbs show promise as early diagnostic markers for Tau-related neurodegeneration.

S-Adenosylhomocysteinase (AHCY, also known as SAHH) is a highly conserved enzyme that catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) into adenosine and homocysteine. As the sole enzyme capable of catalyzing this reaction, AHCY modulates cellular methylation potential required for DNA, RNA, and protein methyltransferase activity. Recent discoveries, however, expand its role well beyond this canonical function, positioning AHCY as a metabolic gatekeeper that integrates one-carbon metabolism with epigenetic regulation, RNA processing, nucleotide balance, and redox signaling. This review brings together mechanistic, structural, and regulatory insights into AHCY while critically evaluating diverse biochemical and biophysical methods for assaying its activity. Comparative structural analyses uncover conserved tetrameric organization alongside species-specific adaptations in oligomeric state, NAD⁺ pocket accessibility, and C-terminal dynamics that shape enzyme catalytic efficiency and regulation. AHCY function is further fine-tuned through a wide spectrum of post-translational modifications and small-molecule interactions, linking it to transcriptional control, stress adaptation, and viral infection. By linking SAH turnover to methylation capacity and adenosine/homocysteine flux, AHCY coordinates metabolism with chromatin regulation and stress responses. These cross-cutting roles highlight how a single metabolic enzyme bridges catalysis, regulation, and disease. In doing so, AHCY exemplifies the broader principle that metabolic enzymes can have a central role as regulators of metabolic flux and cellular regulation, offering both mechanistic depth and translational promise as a therapeutic target.

Early-stage spliceosome assembly is critical to constitutive and alternative pre-mRNA splicing. This process is orchestrated by serine/arginine-rich (SR) proteins (SRSF1-SRSF12) and SR-related proteins U1-70K and U2AF1. SR proteins recognize exonic splicing enhancers and interact with U1-70K and U2AF1 to recruit the U1 and U2 snRNP complexes to the 5' and 3' splice sites, respectively. However, the molecular basis of the interaction between SR proteins and U2AF1 has remained poorly understood, largely due to the poor solubility of full-length U2AF1. Here, we successfully refold and solubilize U2AF1 and confirm its structural integrity. This enables investigation of its interaction with SRSF1, a prototypical SR protein. We show that the U2AF1 C-terminal RS domain (RS^U2AF1) is essential for binding to the phosphorylated RS domain of SRSF1 (RS^SRSF1), and that RS^U2AF1 is phosphorylated in cells. Notably, phosphorylation of RS^U2AF1 significantly reduces its affinity for SRSF1, revealing a phosphorylation-dependent regulatory mechanism. The SRSF1-U2AF1 interaction closely parallels that of SRSF1 and U1-70K, hinting at a general principle in which phosphorylated RS interacts with unphosphorylated ones. Inspired by this discovery, we further find the interaction between phosphorylated and unphosphorylated SRSF1, providing a mechanistic explanation of long observed self-interactions within SR proteins. Our MD simulations further reveal that the salt-bridges between phosphoserine and arginine dominate these interactions, and the interaction strength depends on net charges of RS regions. Together, our findings provide new molecular insights into how phosphorylation modulates splicing factor interactions and highlight a conserved mechanism that regulates early spliceosome assembly.