Journal of biochemistry最新文献

Cellular senescence is a stress-induced, stable growth arrest accompanied by marked metabolic alterations and acquisition of the senescence-associated secretory phenotype (SASP). While enhanced glycolysis, mitochondrial dysfunction, and lysosomal abnormalities are well-established features, emerging evidence identifies progressive intracellular acidification as an important yet underappreciated regulator of cellular senescence. Acidification results from suppressed NHE1-mediated proton efflux, elevated glycolytic proton production, and lysosomal membrane permeabilization. This lowered pH alters redox balance, inhibits HDAC activity, and promotes transcription of senescence-associated genes. Recent work by Kawakami et al. demonstrates that acidification activates a glycolysis-linked inflammatory circuit through accumulation of glucose-6-phosphate and induction of the MondoA targets TXNIP and ARRDC4, which correlate with SASP induction and define a highly secretory subset of senescent cells. These findings suggest that intracellular pH functions as a key metabolic cue linking altered glycolysis to inflammatory output, offering a conceptual framework that may guide future efforts to modulate age-associated chronic inflammation.

LAMP2 is one of the major lysosomal membrane proteins. It contains a large luminal domain, a single transmembrane (TM) domain, and an unusually short cytoplasmic tail composed of only 11 amino acids. Three splicing variants-LAMP-2A, LAMP-2B, and LAMP-2C-share an identical luminal domain but differ in their TM and cytoplasmic tail sequences, resulting in distinct trafficking pathways and functions. Yamaguchi et al. demonstrated that the ultimate target compartments of these isoforms diverge according to the binding affinities of their cytoplasmic tails for μ-subunits of adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4. Intriguingly, each isoform contributes to specific lysosomal functions. It is remarkable that such short cytoplasmic tails not only determine subcellular localization but also underlie the functional diversity of LAMP-2 isoforms.

Mitochondrial quality control plays a critical role in maintaining cellular homeostasis by eliminating dysfunctional mitochondria. The PINK1/Parkin-dependent mitophagy mediates the selective clearance of damaged mitochondria. Dysfunction of PINK1 and Parkin is closely linked to Parkinson's disease. Upon mitochondrial depolarization, PINK1 accumulates on the outer membrane and phosphorylates both ubiquitin and the UBL domain of Parkin to initiate a positive feedback loop of ubiquitination. Parkin catalyzes the assembly of heterogeneous ubiquitin chains on outer mitochondrial membrane proteins, which serve as signals for autophagy adaptors. These adaptors are regulated by kinases such as TANK-binding kinase (TBK1). Deubiquitinating enzymes such as USP30 act as negative regulators. Recent structural and biochemical studies have advanced our understanding of the PINK1/Parkin-dependent mitophagy. Nonetheless, important questions remain regarding the regulatory mechanisms of PINK1, the catalytic mechanism of ubiquitin chain formation by Parkin, and the recognition of ubiquitin chains by autophagy adaptors. Here, we review the current understanding and outstanding questions on the molecular mechanisms underlying the PINK1/Parkin-dependent mitophagy with a focus on ubiquitin signaling.

Ubiquitin modifications are a central hub for numerous biomolecular reactions, finely tuning essential processes such as protein degradation and cellular signal transduction. The architecture of ubiquitin chains-linkage types, branching patterns, and lengths-encodes a rich layer of information, referred to as the ubiquitin code. This intricate code orchestrates diverse biological outcomes. Notably, earlier studies have revealed that branched ubiquitin chains accelerate proteasomal degradation. Yet, the delicate molecular choreography that drives this enhancement remains a mystery, waiting to be fully unraveled. Recent structural, biochemical, and chemical biology approaches have provided new insights into the molecular mechanisms underlying the encoding and decoding of branched ubiquitin chain. In this review, we focus on the proteolytic codes of K11/K48-, K29/K48-, and K48/K63-branched ubiquitin chains, offering an overview of recent research and discussing future challenges and prospects.

Macromolecular crowding is a fundamental property of the intracellular environment that influences protein folding, enzymatic activity, and phase behavior. Disruptions to the homeostasis of macromolecular crowding can drive pathological processes, such as aberrant liquid-liquid phase separation and protein aggregation, which are central features of several neurodegenerative diseases. However, tools for quantifying crowding and aggregation remain limited. Here, we describe moxCRONOS, a Förster resonance energy transfer (FRET)-based biosensor that enables the quantitative measurement of macromolecular crowding and protein condensation. moxCRONOS retains the optical properties of the original CRONOS sensor but offers enhanced stability in oxidative environments, such as within the endoplasmic reticulum or under sodium arsenite treatment, allowing for direct comparison of crowding levels across organelles regardless of redox conditions. Moreover, when fused to dipeptide repeat proteins associated with C9ORF72-linked neurodegeneration, moxCRONOS detects aggregation-prone states-especially in cells expressing glycine-alanine (GA) repeats. Using fluorescence-activated cell sorting, we achieved sensitive and quantitative detection of heterogeneous high-FRET cell populations containing GA aggregates. FRET signal intensity increased upon treatment with a molecular crowding agent or a proteasome inhibitor. These findings establish moxCRONOS as a versatile biosensor for investigating both physiological macromolecular crowding and pathological protein aggregation, with significant potential for disease modeling and therapeutic screening.

Human aldehyde dehydrogenase IV (hALDH4) role in the metabolism of aldehydic compounds is apodictic. Fisetin, a bioactive flavonoid, having myriad of pharmacological activities with inexhaustible therapeutic potentials. Howbeit, the interactive mechanism and inhibitory potential of fisetin on hALDH4 still remain unclear and untold. Here, multi-spectroscopic technique, molecular modelling and dynamic simulations were comprehensively explored to elucidate this. Fisetin quenched the intrinsic fluorescence of the hALDH4 and showed a significant inhibitory effect on the enzyme (IC50 = 17.45 μM) with kinetic inhibition constant, KI, of 25.97 μM. It reversibly inhibited the enzyme in a mixed competitive manner. The interaction, though predominantly electrostatic interaction, perturbed the intrinsic hALDH4 conformation by compromising the predominant α-helix structure. hALDH4 has one ligand competent site for fisetin with a binding constant (Ka) of 3.80 × 104 L·mol-1 at 25°C. The molecular docking and atomistic simulations demonstrated affinity of fisetin for hALDH4 causing the protein structural strain, resulting in unusual but stable conformations. These findings provided important insight into the kinetics and thermodynamics of fisetin and hALDH4 interaction; thus. shedding light on the potential treatment of hALDH-implicated pathological conditions.

The murine retrovirus integration site 1 (MRVI1) gene encodes an endoplasmic reticulum (ER)-associated membrane protein involved in calcium signalling, yet its molecular interaction network remains largely undefined. Here, we employed TurboID-based proximity labelling to construct the first comprehensive map of MRVI1-associated proteins in mammalian cells. This analysis identified >700 candidate interactors, including ER-localized factors and components of intracellular trafficking, consistent with the subcellular localization and signalling role of MRVI1. To investigate oncogenic modulation, we examined how co-expression of NPM-ALK-a constitutively active tyrosine kinase implicated in lymphoid malignancies-reshapes the MRVI1 interactome. Quantitative proteomics revealed that while the overall composition of MRVI1-associated proteins was largely preserved, a subset of interactions was selectively enhanced or attenuated by NPM-ALK. The association of MRVI1 with several signalling-related proteins was enhanced by NPM-ALK, including 12 proteins that have all been previously implicated in cancer-related pathways. In contrast, proteins whose interaction with MRVI1 was suppressed were functionally enriched in the Gene Ontology term 'negative regulation of apoptotic process'. Notably, anti-apoptotic regulators such as DDB1, PHB2 and NOTCH2 showed significantly reduced proximity labelling, suggesting that MRVI1 may participate in apoptosis-related networks disrupted during oncogenic transformation. Together, our findings demonstrate that MRVI1 forms a functionally diverse protein network that can be selectively remodelled by oncogenic signalling. This study not only uncovers potential mechanisms by which MRVI1 contributes to transformation but also provides a valuable proteomic resource for future investigation of MRVI1 function and regulation.