Journal of biochemistry最新文献

Multidrug resistance in Pseudomonas aeruginosa is strongly promoted by the resistance-nodulation-division (RND) family tripartite efflux pump MexAB-OprM, whose inner-membrane transporter MexB plays a central role in recognizing and extruding a broad spectrum of antibiotics and detergents. Although crystal structures of MexB have been determined, no structure of MexB bound to an antibiotic has previously been reported. Here, we report crystal structures of drug-free MexB and chloramphenicol-bound MexB crystallized under mildly basic conditions. In the chloramphenicol-bound structure, chloramphenicol binds at the deep end of the distal binding pocket (DBP) groove in the Binding protomer. Based on this structure, we identified DBP residues (Q125, R128, F178, G179, S180, and Q273) that contact chloramphenicol and evaluated their contributions using in vitro chloramphenicol resistance assays of single-substitution MexB variants. Substitutions at these positions reduced cell growth in the presence of chloramphenicol, minocycline, levofloxacin, and the detergent CYMAL-7. These findings identify a MexB-specific recognition subsite within the DBP groove and provide a structural basis for understanding how MexB recognizes chloramphenicol and other chemically diverse substrates.

Fab has been extensively studied to elucidate the structural and physicochemical principles underlying antigen recognition. While most investigations have focused on IgG-derived Fabs, comparatively little is known about those from other isotypes. To investigate the role of the IgM constant domain Cμ1, we engineered a chimeric Fab (Cμ1Fab) in which the CH1 domain of adalimumab Fab (IgG1) was replaced by the human IgM Cμ1 domain. Cμ1Fab was expressed in CHO cells and its physicochemical properties were evaluated. SDS-PAGE analysis with or without PNGase F treatment demonstrated that the N166-N167-S168 motif was N-glycosylated. SPR analysis demonstrated that Cμ1Fab retained antigen-binding activity comparable to that of the wild-type Fab, and elimination of the glycosylation motif (N166A) did not alter binding affinity. Notably, N-linked glycosylation within the Cμ1 domain suppressed aggregation, whereas the N166A variant showed marked aggregation. DSC further revealed that the glycosylation affected thermal stability. The melting temperature (Tm) of the N166A variant was 60.5 °C, substantially lower than that of the Cμ1Fab (Tm = 66.0 °C), although Tm of the wild-type Fab was 75.2 °C. Collectively, these findings establish the critical role of native Cμ1 N-linked glycosylation in Fab stability, offering new insights for antibody engineering.

Accurate initiation of eukaryotic translation is essential for proteome integrity, yet the process is intrinsically challenged by the potential use of non-AUG start codons. The eIF5-mimic protein 5MP1 (also known as BZW2) has emerged as a pivotal regulator that enhances stringency in start codon selection by competing with eIF5 and eIF2B for binding to eIF2 and eIF3. This competition suppresses non-AUG and repeat-associated non-AUG (RAN) translation and remodels the dynamics of the scanning preinitiation complex. Beyond its biochemical role, accumulating evidence since the first report of BZW1/5MP2 as an oncogene in 2009 indicates that 5MP1/2 proteins promote proliferation, metastasis, and poor prognosis across diverse cancer types. However, the downstream effectors and mechanistic pathways linking translation control to tumorigenesis remain incompletely defined. This review summarizes current biochemical understanding of 5MP1 in translation initiation and synthesizes evidence supporting its tumor-promoting activities, outlining key questions and future directions.

Nonsulfated Human Natural Killer-1 (nsHNK-1) glycan is a trisaccharide biosynthesized by β-1,3-glucuronyltransferase 2 (GlcAT-S) and expressed in the mouse kidney, especially the apical membrane of proximal tubules. Although such apical membrane proteins are known to shed into urine, the presence and significance of urinary nsHNK-1 glycans have remained unexplored. Here, we demonstrated that nsHNK-1 glycan is detectable in mouse urine and identified its major carrier as meprin A subunit alpha (MEP1A), a metalloprotease enriched at the apical membrane of proximal tubules and known to be excreted into urine. Mass spectrometry revealed nsHNK-1 glycosylation at four sites on MEP1A, with the highest occupancy at Asn221 in the catalytic domain. In GlcAT-S knockout mice, urinary MEP1A lacked the nsHNK-1 glycan and exhibited reduced enzymatic activity, suggesting nsHNK-1 glycosylation has a functional contribution. Induced tubular injury decreased urinary MEP1A-associated nsHNK-1 levels, following a similar pattern to reduced MEP1A-associated nsHNK-1 levels in the kidney. In contrast, glomerular injury resulted in increased MEP1A-associated nsHNK-1 expression in the kidney, accompanied by elevated urinary nsHNK-1 levels. These findings suggest that urinary nsHNK-1 levels differentially reflect renal dysfunction and may serve as a novel biomarker of kidney function.

Diabetic foot ulcers (DFUs) are a severe complication of diabetes mellitus, characterized by impaired wound healing due to complex pathophysiological mechanisms. Cellular senescence, particularly the senescence-associated secretory phenotype (SASP), contributes to delayed healing by inducing persistent inflammation and dysfunction in dermal fibroblasts, macrophages, and adipose tissue cells. Here we review the molecular pathways leading to senescence in these cell types, including p53/p21 activation and apoptosis resistance, and how their SASP perpetuates chronic inflammation and impairs tissue regeneration. We also discuss emerging therapeutic approaches targeting senescent cells with senolytic and senomorphic agents to improve healing outcomes. These insights suggest that modulating cellular senescence may offer promising avenues for treating diabetic wounds, warranting further investigation into senescence-targeted therapies in clinical settings.

Because they are continually exposed to fluctuating environments, photosynthetic organisms, including land plants and algae, must adapt to a wide range of environmental conditions. For this purpose, translational control plays a pivotal role. The advent of ribosome profiling, or Ribo-Seq, has helped overcome the technical barriers faced by earlier methods, enabling comprehensive and quantitative analysis of protein synthesis. This review highlights recent advances in green lineage Ribo-Seq, covering technical innovations, diverse applications, and analytical insights. These findings emphasize the power and versatility of this technique in exploring translational regulation in photosynthetic species.

Neprilysin (NEP) is a major enzyme that degrades amyloid β-peptide (Aβ) in the brain. Deficient NEP activity causes Aβ to accumulate, leading to amyloid pathology in Alzheimer's disease. Transcripts from the MME gene encoding NEP comprise seven splice variants (v1, v1bis, v2a, v2b, and v3-5) with different structures in the 5'-untranslated region. We analyzed the expression levels of MME variants in neuronal and non-neuronal cells using real-time PCR and investigated their translational outputs using cells co-transfected with cDNAs of each MME variant and green fluorescent protein. v1 and v1bis were constitutively expressed as major components in both cell types, whereas v2b was preferentially expressed in non-neuronal cells. The translational outputs of v1 and v2b were similar to each other and much higher than those of the other variants. Retinoic acid-induced neuronal differentiation altered the compositional ratios of MME variants and transiently enhanced their translational outputs, which declined in a variant-specific manner with further differentiation. We demonstrated that MME variants show cell type-dependent expression and distinct translational outputs that are sensitive to retinoic acid. Further studies are required to elucidate the molecular mechanisms underlying the post-translational roles of MME variants, such as subcellular localization and local translation.

High-density lipoprotein (HDL) mediates reverse cholesterol transport from the peripheral tissues to the liver and plays a crucial role in preventing atherosclerosis. ATP-binding cassette transporter A1 (ABCA1) is essential for HDL biogenesis; however, whether cholesterol serves as a substrate that is directly transported by this protein remains unclear. Sakata et al. reconstituted ABCA1 into phospholipid liposomes devoid of cholesterol during assembly, enabling an accurate evaluation of substrate specificity. Their findings demonstrated that cholesterol, but not plant sterols, stimulate ABCA1 ATPase activity, indicating that cholesterol is the preferred substrate. Moreover, ABCA1 activity was found to require anionic phospholipids, suggesting that the membrane lipid composition regulates its function. These results provide biochemical evidence of ATP-driven cholesterol transport by ABCA1 and establish a versatile platform for future studies on lipid homeostasis and HDL formation.

The Hippo pathway, traditionally recognized for its tumor suppressive function, has been paradoxically implicated in promoting tumor progression in cancers such as estrogen receptor-positive breast cancer. Treatment of ER-positive breast cancer faces notable challenges owing to the growing incidence of tumor resistance to hormonal therapies, highlighting the need for alternative therapeutic strategies. Here, we explored potential Hippo pathway inhibitors through the multistep screening of 1,150 kinase inhibitors and identified GSK690693 as a potent inhibitor of the Hippo pathway, specifically targeting LATS1 kinase activity. GSK690693 effectively downregulated ER expression in MCF-7 breast cancer cells by suppressing the Hippo pathway activity. Mechanistically, treatment with GSK690693 led to cell cycle arrest and induction of cell death. These findings revealed a previously undefined mechanism of action of GSK690693 as a Hippo pathway inhibitor, underscoring its efficacy in mitigating ER-positive breast cancer progression. Given the broader implications of Hippo pathway dysregulation in multiple cancers, GSK690693 could be part of a combination regimen for various malignancies.

Tetrahydrobiopterin (BH4) is an essential cofactor for biosynthesis of monoamines and nitric oxide. An excess of BH4 in infiltrated macrophages was reported to cause pain, while a certain level of BH4 is essential for cell survival and proliferation. GTP cyclohydrolase I (GCH) is a rate-limiting enzyme for the de novo synthesis of BH4. Our previous study showed that GCH expression was elevated by an enhancer region containing the C/EBP and Ets binding motifs in macrophage-like RAW264.7 cells when stimulated with lipopolysaccharide (LPS). In this study, we showed that poly(I:C) and R848, Toll-like receptors ligands for RNA viruses, increased GCH expression and BH4 levels in RAW264.7 cells as well as bacterial LPS. We examined the intracellular signaling pathway for the induction of the Gch gene, and found that inhibitors for the NF-κB pathway suppressed the GCH expression by these stimuli. We for the first time identified the region required for LPS-induced GCH expression to be the 5'-untranslted region of exon 1 consisting of 149 bp using a reporter experiment. We also demonstrated that the expression of GCH with LPS was strongly suppressed by an inhibitor of NF-κB in mouse intraperitoneal macrophages in vivo.