Zinc transporter 1 (ZNT1) expression on the cell surface is elaborately controlled by cellular zinc levels

Zinc transporter 1 (ZNT1) is the only zinc transporter predominantly located on the plasma membrane, where it plays a pivotal role exporting cytosolic zinc to the extracellular space. Numerous studies have focused on the physiological and pathological functions of ZNT1. However, its biochemical features remain poorly understood. Here, we investigated the regulation of ZNT1 expression in human and vertebrate cells, and found that ZNT1 expression is posttranslationally regulated by cellular zinc status. We observed that under zinc-sufficient conditions, ZNT1 accumulates on the plasma membrane, consistent with its zinc efflux function. In contrast, under zinc-deficient conditions, ZNT1 molecules on the plasma membrane were endocytosed and degraded through both the proteasomal and lysosomal pathways. Zinc-responsive ZNT1 expression corresponded with that of metallothionein, supporting the idea that ZNT1 and metallothionein cooperatively regulate cellular zinc homeostasis. ZNT1 is N-glycosylated on Asn299 in the extracellular loop between transmembrane domains V and VI, and this appears to be involved in the regulation of ZNT1 stability, as nonglycosylated ZNT1 is more stable. However, this posttranslational modification had no effect on ZNT1's ability to confer cellular resistance against high zinc levels or its subcellular localization. Our results provide molecular insights into ZNT1-mediated regulation of cellular zinc homeostasis, and indicate that the control of cellular and systemic zinc homeostasis via dynamic regulation of ZNT1 expression is more sophisticated than previously thought

Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B″ (δ) regulatory subunit in vitro and identification of the phosphorylation sites

AbstractHuman erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B″ (δ) subunit, was phosphorylated at serine residues of B″ in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 μM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B″, respectively. The Km value of A-kinase for CAB″ was 0.17±0.01 μM in the presence of OA. The major in vitro phosphorylation sites of B″ were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B″ did not dissociate B″ from CA, and stimulated the molecular activity of CAB″ toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a

Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data

<p>Abstract</p> <p>Background</p> <p><it>Bacillus subtilis </it>natto is closely related to the laboratory standard strain <it>B. subtilis </it>Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated <it>B. subtilis </it>168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, <it>B. subtilis </it>natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length.</p> <p>Results</p> <p>We applied a comparative genome assembly method, which combines <it>de novo </it>assembly and reference guided assembly, to one of the <it>B. subtilis </it>natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for <it>B. subtilis </it>natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of <it>degQ </it>and the coding region of <it>swrAA </it>as the wild strain, RO-FF-1.</p> <p>These are specific for γ-PGA production ability, which is related to natto production. Further, the <it>B. subtilis </it>natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases.</p> <p>Conclusions</p> <p>The determination of the whole genome sequence of <it>Bacillus subtilis </it>natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that <it>B. subtilis </it>natto harbors but <it>B. subtilis </it>168 lacks. Multiple genome-level comparisons among five closely related <it>Bacillus </it>species were also carried out. The determined genome sequence of <it>B. subtilis </it>natto and gene annotations are available from the Natto genome browser <url>http://natto-genome.org/</url>.</p

Pigmentation and TYRP1 expression are mediated by zinc through the early secretory pathway-resident ZNT proteins

メラニン生合成には、亜鉛も必要だった！. 京都大学プレスリリース. 2023-04-19.Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are essential for pigmentation. They are generally classified as type-3 copper proteins, with binuclear copper active sites. Although there is experimental evidence for a copper cofactor in TYR, delivered via the copper transporter, ATP7A, the presence of copper in TYRP1 and TYRP2 has not been demonstrated. Here, we report that the expression and function of TYRP1 requires zinc, mediated by ZNT5–ZNT6 heterodimers (ZNT5–6) or ZNT7–ZNT7 homodimers (ZNT7). Loss of ZNT5–6 and ZNT7 function results in hypopigmentation in medaka fish and human melanoma cells, and is accompanied by immature melanosomes and reduced melanin content, as observed in TYRP1 dysfunction. The requirement of ZNT5–6 and ZNT7 for TYRP1 expression is conserved in human, mouse, and chicken orthologs. Our results provide novel insights into the pigmentation process and address questions regarding metalation in tyrosinase protein family

Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 as a Manganese Transporter

Manganese (Mn) homeostasis involves coordinated regulation of specific proteins involved in Mn influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved, nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in Mn detoxification/efflux, by evaluating their ability to reduce Mn toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and a zinc-specific transporter hZnT1 showed that residue N43, which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique Mn mobilization activity; residues C52 and L242 in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the H->N reversion mutant in hZnT1 conferred Mn transport activity and loss of zinc transport activity. These results provide important information about Mn detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a Mn transporter

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway

Group IIA secreted phospholipase A2 controls skin carcinogenesis and psoriasis by shaping the gut microbiota

Besides promoting inflammation by mobilizing lipid mediators, group IIA secreted phospholipase A2 (sPLA2-IIA) prevents bacterial infection by degrading bacterial membranes. Here, we show that, despite the restricted intestinal expression of sPLA2-IIA in BALB/c mice, its genetic deletion leads to amelioration of cancer and exacerbation of psoriasis in distal skin. Intestinal expression of sPLA2-IIA is reduced after treatment with antibiotics or under germ-free conditions, suggesting its upregulation by gut microbiota. Metagenome, transcriptome, and metabolome analyses have revealed that sPLA2-IIA deficiency alters the gut microbiota, accompanied by notable changes in the intestinal expression of genes related to immunity and metabolism, as well as in the levels of various blood metabolites and fecal bacterial lipids, suggesting that sPLA2-IIA contributes to shaping of the gut microbiota. The skin phenotypes in Pla2g2a–/– mice are lost (a) when they are cohoused with littermate WT mice, resulting in the mixing of the microbiota between the genotypes, or (b) when they are housed in a more stringent pathogen-free facility, where Pla2g2a expression in WT mice is low and the gut microbial compositions in both genotypes are nearly identical. Thus, our results highlight a potentially new aspect of sPLA2-IIA as a modulator of gut microbiota, perturbation of which affects distal skin responses

Group III phospholipase A2 promotes colitis and colorectal cancer

Lipid mediators play pivotal roles in colorectal cancer and colitis, but only a limited member of the phospholipase A2 (PLA2) subtypes, which lie upstream of various lipid mediators, have been implicated in the positive or negative regulation of these diseases. Clinical and biochemical evidence suggests that secreted PLA2 group III (sPLA2-III) is associated with colorectal cancer, although its precise role remains obscure. Here we have found that sPLA2-III-null (Pla2g3−/−) mice are highly resistant to colon carcinogenesis. Furthermore, Pla2g3−/− mice are less susceptible to dextran sulfate-induced colitis, implying that the amelioration of colonic inflammation by sPLA2-III ablation may underlie the protective effect against colon cancer. Lipidomics analysis of the colon revealed significant reduction of pro-inflammatory/pro-tumorigenic lysophosholipids as well as unusual steady-state elevation of colon-protective fatty acids and their oxygenated metabolites in Pla2g3−/− mice. Overall, our results establish a role of sPLA2-III in the promotion of colorectal inflammation and cancer, expand our understanding of the divergent roles of multiple PLA2 enzymes in the gastrointestinal tract, and point to sPLA2-III as a novel druggable target for colorectal diseases

Circadian protection against bacterial skin infection by epidermal CXCL14-mediated innate immunity

体内時計は夜間に自然免疫を発動 --皮膚ケモカインによる自然免疫機構--. 京都大学プレスリリース. 2022-06-16.Biological clocks set for skin immunity. 京都大学プレスリリース. 2022-06-21.The epidermis is the outermost layer of the skin and the body’s primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system

Foxn1−β5t転写制御軸は胸腺でのCD8陽性T細胞生成を制御する

The thymus is an organ that produces functionally competent T cells that protect us from pathogens and malignancies. Foxn1 is a transcription factor that is essential for thymus organogenesis; however, the direct target for Foxn1 to actuate thymic T-cell production is unknown. Here we show that a Foxn1-binding cis-regulatory element promotes the transcription of β5t, which has an essential role in cortical thymic epithelial cells to induce positive selection of functionally competent CD8+ T cells. A point mutation in this genome element results in a defect in β5t expression and CD8+ T-cell production in mice. The results reveal a Foxn1-β5t transcriptional axis that governs CD8+ T-cell production in the thymus