Journal of Biological Chemistry最新文献

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.

Alginate lyases (ALs) cleave 4-O-glycosidic linkages in alginate, composed of mannuronate (M) and guluronate (G) residues via β-elimination with preference for either one or several M-M, M-G, G-M, G-G linkages. ALs in polysaccharide lyase family 6 (PL6), present different specificities and modes of action, contain either one or two parallel β-helix domains. About half of almost 600 PL6 sequences are of the two-domain type, all located in the phyla Pseudomonadota and Bacteroidota. Here, functional roles are described for the N- and C-terminal domains (NTD and CTD) using BoPL6, a two-domain AL from the human gut bacterium Bacteroides ovatus CP926, which is specific for G in subsite +1. The NTD contains the catalytic site, but BoPL6-NTD markedly preferred the model substrate polyMG and cleaved M-G bonds in endo-mode, whereas the NTD + CTD mixture, similarly to BoPL6, acted with highest activity on model substrate polyG in exo-mode, verified by time-resolved ¹H-NMR. CTD was not catalytically active but bound polyguluronate and, when mixed with BoPL6-NTD, promoted activity on polyG, yielding products of DP 1‒3, similarly to BoPL6. This defines a crucial role of CTD in shaping the active site in BoPL6, as validated by substrate docking. The BoPL6 E634A mutant in the conserved CTD DEST loop, interacting with the active site in two-domain PL6 enzymes, was inactive, while the corresponding CTD mutant mixed with the NTD did not form the WT structure and had highly reduced activity on polyG, but acted on polyMG in endo-mode with improved rate and conversion.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone widely used to manage diabetes and obesity, through its ability to regulate glucose homeostasis. Clinically relevant GLP-1 sequences form oligomeric states. Uncontrolled oligomer formation can drive fibril formation, posing challenges such as difficulty in controlling drug dosage, loss of activity, or toxicity, as the aggregates can be immunogenic and/or can form amyloids. Here, we used combined measurements of colloidal and conformational stability to characterise the intermolecular interactions underpinning the physical status of the GLP-1 7-37 amide (GLP-1am), at pharmaceutically relevant high concentrations. We focus on less explored conditions, around pH 5, mimicking the environment within native cellular secretory granules, where the hormone is also densely packed. Co-solutes allowed us to interfere with weak interactions affecting peptide self-association into soluble oligomers, and the conversion into aggregates and fibrils. We show that GLP-1am exists as soluble oligomers that assemble into nanosheets over the timescale of hours, in quiescent conditions. Aggregation proceeded via a nucleation-dependent mechanism, with its rate correlating to the magnitude of attractive intermolecular interactions. It was accelerated by ionic co-solutes, indicating a key role for screening of electrostatic interactions in modulating peptide-peptide attraction and assembly. The rate of aggregation was also pH-dependent, with rates being slower at pH 5 than pH 8. Notably, the addition of proline, as a co-solute, delayed the onset of GLP-1am aggregation in a pH-dependent manner. Thus, in quiescent conditions, GLP-1am forms discrete soluble oligomers capable of organising into ordered nanostructures rather than amyloid fibrils.

Electromotility in mammalian outer hair cells (OHC) is the mechanism underlying cochlear amplification. It is brought about by the piezoelectric-like property of the membrane protein prestin (Slc26a5) that lies in the OHCs lateral plasma membrane. Prestin connects to an underlying cytoskeletal network of circumferential actin filaments that bridge longitudinal spectrin filaments. This network, in turn, lies between the plasma membrane and a closely apposed ER-like tubular array of subsurface cisternae (SSC). Two previous papers examining spectrin knockouts in embryonic hair cells were confined to analyzing the effects on the apical cuticular plate and overlying stereocilia. In this paper, we examine the effects of conditional knockouts of alpha2 spectrin in postnatal OHCs. We find a significant auditory phenotype likely due to the novel disassociation of prestin's gating charge movement from OHC electromotility. In addition, OHCs show enlargement in their SSC and plasma membrane-SSC space with preserved cuticular plates and overlying stereocilia, the latter contrasts with the findings in embryonic knockouts.

The small GTPase Rab9 plays a major role in the vesicular trafficking of mannose-6-phosphate receptor (CI-M6PR). CI-M6PR trafficking has also been reported to be perturbed by dysfunction of a ubiquitin ligase necessary for protein quality control (PQC). However, the mechanism underlying the participation of the PQC machinery in CI-M6PR trafficking is poorly understood. In this study, we found an extremely short half-life of GDP-bound Rab9, which is in clear contrast to its phylogenetically closest relative, Rab7. Comparison of the amino acid sequences of these relatives revealed that hydrophobic residues are specifically exposed in the Switch I region of Rab9a and that these residues are recognized by the PQC machinery. We defined this exposed hydrophobicity as a conformation-dependent hydrophobic (CDH) degron because its existence determines the instability of Rab proteins in a nucleotide-dependent manner. CDH degron-mediated instability is essential for Rab9a function, given that forced accumulation of CDH degron-mutated Rab9a in cells resulted in the defective localization of CI-M6PR, a similar phenotype observed in PQC dysfunction. Thus, the CDH degron-driven PQC system is necessary for the proper vesicular trafficking of CI-M6PR. We also identified VCP/p97 as a CDH degron-dependent PQC factor for GDP-bound Rab9a.

Myotonic Dystrophy type 1 (DM1) is caused by a CUG expansion located in the 3' untranslated region (UTR) of dystrophia myotonica protein kinase (DMPK) mRNAs. The pathogenic model underlying DM1 implicates the accumulation of mutant DMPK transcripts in nuclei where they form toxic RNA foci. This, in turn, disrupts the functional availability of several RNA-binding proteins involved in pre-mRNA alternative splicing. Consequently, such dysregulations result in widespread missplicing of multiple mRNAs accounting for the plethora of DM1 symptoms. Accordingly, DM1 is referred to as a spliceopathy. Over the years, multiple signaling pathways have also been reported to be disrupted in DM1, especially in skeletal muscle. In this review, we focus on several of these pathways including protein kinase R (PKR), protein kinase C (PKC), glycogen synthase kinase 3β (GSK-3β), Akt-mTOR, AMP-activated protein kinase (AMPK), TWEAK-Fn14 and NF-kB, and calcineurin-NFAT. We describe the individual effects of these signaling disruptions on multiple skeletal muscle functions and characteristics, and we also present an overview of their cumulative impact. Based on the available literature, the dysregulation of signaling in skeletal muscle jointly results in global perturbations in protein synthesis and degradation, muscle repair, mitochondrial biogenesis, energy metabolism and inflammation. Despite these advances, the full spectrum of alterations in signaling pathways in DM1 muscle remains incomplete and a certain level of variability in the extent of signaling defects in DM1 muscles has been observed likely due to varied experimental approaches and designs. Additional key unanswered questions relate to how mechanistically the CUG expansion in DMPK mRNAs causes the dysregulation of multiple signaling cascades, and whether missplicing of pivotal signaling molecules within these various pathways further contributes to signaling defects. The fact that pharmacological, physiological, and transgenic approaches targeting these pathways have corrected defects observed in DM1 muscle provides a strong rationale for therapeutic intervention. These pathways can be targeted either individually or through combinatorial treatments involving two or more agents and/or strategies. Based on the importance and impact of these signaling pathways on multiple aspects of the DM1 muscle phenotype, therapeutically targeting these disruptions is becoming increasingly attractive and represents a critical area for additional research in the quest to slow or reverse muscle dysfunction in DM1.