Journal of biochemistry最新文献

Despite being a carcinogen, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits metastatic melanoma growth by downregulating the signal transducer and activator of transcription 3. However, the molecular mechanisms remain unclear. The aim of this study was to identify tyrosine phosphatases that are involved in TPA-induced inhibition of cell proliferation in metastatic melanoma cells. We screened protein tyrosine phosphatases (PTPs) required for TPA-mediated inhibition of cell proliferation. We identified two PTPs, SH2 domain-containing PTP2 (SH-PTP2/PTPN11) and T-cell PTP (TC-PTP/PTPN2) that play key roles in TPA-mediated inhibition of metastatic melanoma cell growth. Transient expression of SH-PTP2 and TC-PTP induced G0/G1 cell cycle arrest in a phosphatase-dependent manner. Furthermore, SH-PTP2 was translocated to the cell membrane upon TPA treatment, resulting in a decrease in Janus kinase 2 activity. TC-PTP is localized in the nucleus together with the adapter protein ubiquitin-like protein 4A; TC-PTP was translocated to the nuclear periphery upon TPA stimulation. These two signaling pathways, involving SH-PTP2 and TC-PTP, are distinct from those observed in normal melanocytes and benign melanoma cells. These pathways represent previously unknown responses to TPA specific to metastatic melanoma cells. Overall, these findings may contribute to the development of new anticancer agents.

Ectodomain shedding (shedding) is a processing mechanism that cleaves the juxtamembrane region of membrane proteins and solubilizes almost the entire extracellular domain. Shedding irreversibly regulates the localization and function of membrane proteins; however, its physiological role is not fully understood. Previously, we showed that the shedding susceptibility of multiple membrane proteins is altered by skipping or inclusion of skipping exon(s) that encode their juxtamembrane region. In this study, we screened the skipping exon encoding the juxtamembrane region of membrane proteins and found that the shedding susceptibility of UNC5B, a Netrin-1 receptor, is altered by skipping or inclusion of the skipping exon encoding its juxtamembrane region. These results raise the possibility that the biological phenomena involving UNC5B, including neural circuit formation, angiogenesis and cancer development, are regulated by shedding in a splice isoform-dependent manner.

Previous studies have reported several O-linked N-acetylglucosamine (O-GlcNAc) modifications of core histones H2A, H2B, H3 and H4. In parallel, the characteristics and functions of O-GlcNAcylated histones are also shown, and they are involved in various cellular processes, such as development and tumorigenesis, indicating that the exploration of new histone O-GlcNAcylation contributes significantly to the elucidation of molecular mechanisms occurring in cells. Here, we report that O-GlcNAcylation occurs at threonine 71 of histone H4 (H4T71Gc) by developing a monoclonal antibody that recognizes the O-GlcNAcylated H4T71 peptide. Threonine 71 of histone H4 is highly conserved from metazoans to mammals, and H4T71Gc can be detected. Chromatin immunoprecipitation-seq and biochemical analysis revealed that H4T71Gc was localized to the region where histone H3 modified by trimethylation of lysine 9 (H3K9me3) was enriched in a genome-wide manner. H3K9me3 is known to function in chromatin condensation, suggesting that H4T71Gc plays a role in both the progression and maintenance of condensed chromatin in several species.

Cholesterol is a crucial lipid that lowers the phase transition temperature of phospholipid membranes and enhances their stability. Artificial cells with diverse functionalities have been developed by encapsulating transcription-translation reactions within liposomes, with the expectation that cholesterol would similarly contribute to the stabilization of membrane compartments in these artificial cells. In this study, we examined whether cholesterol influences the efficiency of reactions within liposomes. Our results demonstrated that the efficiency of transcription-translation reactions decreases in liposomes containing 40 mol% cholesterol, a level comparable to that of the outer leaflet of the human cell membrane. Furthermore, this decrease in reaction efficiency was found to be independent of liposome size or the efficiency of molecule encapsulation. This study highlights the critical role of cholesterol content in the design of artificial cells and drug delivery systems via liposome fusion, emphasizing the need for careful optimization.

Brown adipocytes dissipate chemical energy as heat and confer protection against type 2 diabetes and obesity. Nuclear factor I-A (NFIA) is a transcription factor that orchestrates the brown fat gene programme by activating cell type-specific enhancers and facilitating the genomic binding of PPARγ, the master regulator of adipogenesis, to these enhancers. NFIA promotes mitochondrial oxidative phosphorylation and thermogenesis, while reciprocally suppressing adipose tissue inflammation, thereby contributing to the maintenance of glucose and body weight homeostasis in mice. Here the author provides an overview of the identification of NFIA as a pivotal regulator of brown adipocyte biology, elucidates its underlying mechanisms of action, examines its implications for systemic metabolism and outlines future perspectives for research in this field.

In higher plants, ferredoxin (Fd) and Fd-NADP+ reductase (FNR) are each present as distinct isoproteins of photosynthetic type and non-photosynthetic type, which exhibit differential function despite their similarity in the 3D structures. In this study, we addressed differential regulation of Fd/FNR reaction between the two types from two perspectives and investigated the amino acid residues of Fd responsible for the differences. Firstly, pH-dependent profile of Fd/FNR electron transfer activity varied among the combinations of the two types of Fd and FNR; non-photosynthetic type FNR showed similar pattern for the two types of Fds while photosynthetic type FNR was previously shown to exhibit opposite pattern, which was explained by the different pH-dependent profile of Km for the two Fds. Secondly, the extent of the suppression of the affinity (in terms of Km value) between Fd and FNR by NADPH significantly varied among the combinations of the two types of Fd:FNR. The difference was shown to be mainly due to the different property of Fd between the two types. Kinetic analyses using site-directed mutants of Fd showed the contribution of C-terminal residues, together with that of 78th residue of Fd, on the differential profile of Fd/FNR reaction by pH and NADPH.

The α-amylase from Klebsiella pneumoniae (KpAmy13), which belongs to glycoside hydrolase family 13 subfamily 19, produces maltohexaose as an initial product when acting on starch and has been characterized as a maltohexaose-producing α-amylase. The crystal structure of KpAmy13 was determined at a resolution of 1.9 Å, revealing the structures of all its domains: N, A, B and C. Domain N resembles the starch-binding domain known as carbohydrate-binding module family 69, found in α-glucan-related proteins. Although domain N does not conserve the starch-binding residues observed in other proteins, it has several hydrophobic residues on its surface, which might be involved in promoting catalysis. The catalytic cleft is located at the bottom of a circular depression. The domain N-truncated mutant of KpAmy13 in complex with maltohexaose showed that its non-reducing end glucose docks at subsite -6. The long and complex structure of domain B contributes to forming a cleft of the right size for six glucose moieties, demonstrating the exo-acting mechanism.

Structural variations of N-glycans critically regulate glycoprotein functions and are involved in various human diseases. N-Acetylglucosaminyltransferase-III (GnT-III or MGAT3) is highly expressed in the brain and kidney and is an N-glycan branching enzyme that biosynthesizes the unique N-glycan branch designated as bisecting GlcNAc. Its roles in Alzheimer's disease and cancer have been revealed, but the functions of bisecting GlcNAc in the kidney are poorly understood. Here, we show that kidneys in the GnT-III-knockout (KO) mouse exhibit impaired body fluid balance and present interstitial edema. To understand the molecular mechanisms further, we biochemically purified the glycoproteins modified by GnT-III in the mouse kidney and identified these proteins using proteomics. We found that the proteins involved in the pathway for angiotensin II (Ang II) metabolism are modified by GnT-III, and that the subcellular localization of angiotensin-converting enzyme was altered in GnT-III-KO cells. Furthermore, the pathology in models of Ang II-related disease was slightly more severe in GnT-III-KO than in wild-type mice. Our data indicate a protective role for bisecting GlcNAc in the mouse kidney, highlighting a newly described link between specific N-glycan structures and renal functions.

Glucuronyltransferase GlcAT-P is a rate-limiting enzyme involved in the biosynthesis of the Human Natural Killer-1 carbohydrate and is essential for acquiring higher brain functions. Alternative splicing produces two isoforms, short-form GlcAT-P (sGlcAT-P) and long-form GlcAT-P (lGlcAT-P), which share identical peptide sequences except for an additional 13 amino acids (AA) in the cytoplasmic N-terminal tail of lGlcAT-P. Although sGlcAT-P localizes to the Golgi apparatus (GA), where many glycosyltransferases reside, lGlcAT-P is distributed in both the GA and endoplasmic reticulum (ER). However, the mechanisms responsible for this distinct intracellular distribution remain poorly understood. In this study, we explored the role of the 13 AA in the cytoplasmic N-tail of lGlcAT-P in trafficking between the GA and the ER using the Retention Using Selective Hooks system. Our findings revealed that lGlcAT-P undergoes enhanced retrograde trafficking from the GA to the ER, whereas its anterograde trafficking from the ER to the GA remains largely unaffected. In addition, three glutamic acid residues within the 13 AA of lGlcAT-P were identified as crucial for promoting retrograde trafficking. These results suggest that the ER distribution of lGlcAT-P is primarily governed by Golgi-to-ER trafficking regulated by specific sequences in its cytoplasmic N-tail.

Skeletal muscle myosin is generally considered insoluble under physiological, low ionic strength, or salt-free conditions due to its tendency to self-assemble into filamentous polymers in vitro. Our previous study showed that myosin can be solubilized in low ionic strength solutions containing l-histidine. However, another report suggested that 1-methylhistidine could not solubilize myosin, and the factors essential for myosin solubilization remain unclear. To elucidate the role of l-histidine in the water solubilization of myosin, we examined myosin solubility and the molecular properties of its rod domain, l-meromyosin, using structurally related buffer compounds. Under salt-free conditions, solubility depended heavily on the acid dissociation constant of buffer, indicating that maintaining a neutral pH is critical. The rod domain showed molecular elongation regardless of the buffer type, yet surface charge and hydrophobicity remained comparable to conditions with high ionic strength. These results suggest that myosin is inherently soluble and maintains its structural integrity under neutral, salt-free conditions. The apparent insolubility under such conditions is likely to result from hydrochloric acid used for pH adjustment. Since l-histidine and imidazole achieve neutrality without acid addition, they are ideal buffers for myosin solubilization.