Biochimica et biophysica acta. General subjects最新文献

{"title":"Characterisation of biocondensate microfluidic flow using array-detector FCS","authors":"Stijn Dilissen ,&nbsp;Pedro L. Silva ,&nbsp;Anastasia Smolentseva ,&nbsp;Tom Kache ,&nbsp;Ronald Thoelen ,&nbsp;Jelle Hendrix","doi":"10.1016/j.bbagen.2024.130673","DOIUrl":"10.1016/j.bbagen.2024.130673","url":null,"abstract":"<div><h3>Background</h3><p>Biomolecular condensation via liquid-liquid phase separation (LLPS) is crucial for orchestrating cellular activities temporospatially. Although the rheological heterogeneity of biocondensates and the structural dynamics of their constituents carry critical functional information, methods to quantitatively study biocondensates are lacking. Single-molecule fluorescence research can offer insights into biocondensation mechanisms. Unfortunately, as dense condensates tend to sink inside their dilute aqueous surroundings, studying their properties via methods relying on Brownian diffusion may fail.</p></div><div><h3>Methods</h3><p>We take a first step towards single-molecule research on condensates of Tau protein under flow in a microfluidic channel of an in-house developed microfluidic chip. Fluorescence correlation spectroscopy (FCS), a well-known technique to collect molecular characteristics within a sample, was employed with a newly commercialised technology, where FCS is performed on an array detector (AD-FCS), providing detailed diffusion and flow information.</p></div><div><h3>Results</h3><p>The AD-FCS technology allowed characterising our microfluidic chip, revealing 3D flow profiles. Subsequently, AD-FCS allowed mapping the flow of Tau condensates while measuring their burst durations through the stationary laser. Lastly, AD-FCS allowed obtaining flow velocity and burst duration data, the latter of which was used to estimate the condensate size distribution within LLPS samples.</p></div><div><h3>Conclusion</h3><p>Studying biocondensates under flow through AD-FCS is promising for single-molecule experiments. In addition, AD-FCS shows its ability to estimate the size distribution in condensate samples in a convenient manner, prompting a new way of investigating biocondensate phase diagrams.</p></div><div><h3>General significance</h3><p>We show that AD-FCS is a valuable tool for advancing research on understanding and characterising LLPS properties of biocondensates.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0304416524001168/pdfft?md5=5cba7af83e947409eb66fa929cefd684&pid=1-s2.0-S0304416524001168-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141726819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Shotgun proteomics of thyroid carcinoma exosomes – Insight into the role of exosomal proteins in carcinogenesis and thyroid homeostasis","authors":"Magdalena Surman ,&nbsp;Magdalena Wilczak ,&nbsp;Urszula Jankowska ,&nbsp;Bożena Skupień-Rabian ,&nbsp;Małgorzata Przybyło","doi":"10.1016/j.bbagen.2024.130672","DOIUrl":"10.1016/j.bbagen.2024.130672","url":null,"abstract":"<div><h3>Background</h3><p>Transport of molecules via exosomes is one of the factors involved in thyroid cancer development, and transported molecules may serve as cancer biomarkers. The aim of the study was to characterize protein content of thyroid-derived exosomes and their functional effect exerted on recipient cells.</p></div><div><h3>Methods</h3><p>LC-MS/MS proteomics of exosomes released by FTC and 8305C thyroid carcinoma cell lines, and Nthy-ori 3–1 normal thyroid follicular cells was performed, followed by bioinformatic analysis and functional tests (wound healing and Alamar Blue assays).</p></div><div><h3>Results</h3><p>Exosomes from Nthy-ori 3–1 cells had the highest number of 1504 proteins, while in exosomes from thyroid carcinoma FTC and 8305C cells 730 and 1304 proteins were identified, respectively. For proteins uniquely found in FTC- and 8305C-derived exosomes, enriched cancer-related gene ontology categories included cell adhesion, positive regulation of cell migration, N-glycosylation, drug resistance, and response to NK/T cell cytotoxicity. Furthermore, through label-free quantification (that identified differentially expressed proteins) and comparison with The Human Protein Atlas database several potential diagnostic and/or prognostic biomarkers were indicated. Finally, exosomes from FTC and 8305C cells displayed ability to stimulate migratory properties of recipient Nthy-ori 3–1 cells. Additionally, 8305C-derived exosomes increased recipient cell viability.</p></div><div><h3>Conclusions</h3><p>Multiple proteins identified in thyroid cancer-derived exosomes have a direct link to thyroid cancer progression. Also, in functional tests exosomes enhanced growth and dissemination of non-transformed thyroid cells.</p></div><div><h3>General significance</h3><p>The obtained results expands the knowledge concerning the role of exosomal proteins in thyroid cancer and indicate potential biomarkers for further evaluation in clinical settings.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141722929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic study of inhibitory peptides with SHP-1 in hypertonic environment for infection model","authors":"Shweta Khandibharad,&nbsp;Shailza Singh","doi":"10.1016/j.bbagen.2024.130670","DOIUrl":"10.1016/j.bbagen.2024.130670","url":null,"abstract":"<div><p>Cutaneous Leishmaniasis, an infectious disease is globally the most prevalent form of leishmaniasis accounting for approximately 1 million cases every year as per world health organization. Infected individuals develop skin lesion which has been reported to be infiltrated by immune cells and parasite with high sodium accumulation creating hypertonic environment. In our work, we tried to mimic the hypertonic environment in virtual environment to study dynamicity of SHP-1 and NFAT5 along with their interactions through molecular dynamics simulation. We validated the SHP-1 and NFAT5 dynamics in infection and HSD conditions to study the impact of hypertonicity derived NFAT5 mediated response to <em>L.major</em> infection. We also evaluated our therapeutic peptides for their binding to SHP-1 and to form stable complex. Membrane stability with the peptides was analyzed to understand their ability to sustain mammalian membrane. We identified PepA to be a potential candidate to interact with SHP-1. Inhibition of SHP-1 through PepA to discern IL-10 and IL-12 reciprocity may be assessed in future and furnish us with a potential therapeutic molecule. HSD mice exhibited high pro-inflammatory response to <em>L.major</em> infection which resulted in reduced lesion size. Contrary to observations in HSD mice, infection model exhibited low pro-inflammatory response and increased lesion size with high parasite load. Thus, increase in NFAT5 expression and reduced SHP-1 expression may result in disease resolving effect which can be further studied through incorporation of synthetic circuit using PepA to modulate IL-10 and IL-12 reciprocity.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141598336","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway","authors":"","doi":"10.1016/j.bbagen.2024.130669","DOIUrl":"10.1016/j.bbagen.2024.130669","url":null,"abstract":"<div><h3>Background</h3><p>Dysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity.</p></div><div><h3>Methods</h3><p>The biological function of ROCK1 was analyzed <em>in vitro</em> by establishing a silenced cell model. Coimmunoprecipitation confirmed the direct binding between ROCK1 and c-MYC, and a luciferase reporter assay revealed the binding of c-MYC to the promoter of the PFKFB3 gene. These results were verified in animal experiments.</p></div><div><h3>Results</h3><p>ROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with a poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased gemcitabine (GEM) sensitivity <em>in vivo</em> and <em>in vitro</em>.</p></div><div><h3>Conclusions</h3><p>ROCK1 promotes glycolytic activity in PC cells and promotes PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth <em>in vivo</em> and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141598337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteolytic cleavage of Golgi glycosyltransferases by SPPL3 and other proteases and its implications for cellular glycosylation","authors":"","doi":"10.1016/j.bbagen.2024.130668","DOIUrl":"10.1016/j.bbagen.2024.130668","url":null,"abstract":"<div><p>Glycosylation of proteins and lipids is of fundamental importance in multicellular eukaryotes. The vast diversity of glycan structures observed is generated in the Golgi apparatus by the concerted activity of &gt;100 distinct enzymes, which include glycosyltransferases and other glycan-modifying enzymes. Well-known for decades, the majority of these enzymes is released from the Golgi apparatus and subsequently secreted into the extracellular space following endoproteolytic cleavage, but the underlying molecular mechanisms and the physiological implications have remained unexplored. This review will summarize our current knowledge of Golgi enzyme proteolysis and secretion and will discuss its conceptual implications for the regulation of cellular glycosylation and the organization of the Golgi apparatus. A particular focus will lie on the intramembrane protease SPPL3, which recently emerged as key protease facilitating Golgi enzyme release and has since been shown to affect a multitude of glycosylation-dependent physiological processes.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0304416524001119/pdfft?md5=b7b7465eeeecf08fe5f868ebd5448820&pid=1-s2.0-S0304416524001119-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141589463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lectins of the Araceae family: Insights, distinctions, and future avenues—A three-decade investigation","authors":"Emadeldin Hassan E. Konozy ,&nbsp;Amina I. Dirar ,&nbsp;Makarim Elfadil M. Osman","doi":"10.1016/j.bbagen.2024.130667","DOIUrl":"10.1016/j.bbagen.2024.130667","url":null,"abstract":"<div><p>The Araceae family boasts &gt;3000 species of flowering plants that thrive across the tropics. Among the focal points of study within this family are lectins, proteins with affinity for binding carbohydrates. This review endeavors to gather data gleaned from numerous studies conducted over the past three decades on lectins extracted from <em>Araceae</em> plants. Our examination spans their extraction and purification methods, their specific interactions with carbohydrates, their molecular structures, and various physicochemical characteristics. Furthermore, we investigated the biological activities of these lectins and investigated the outcomes of cloning their genes. Despite their apparent similarities, these lectins exhibit notable distinctions, particularly regarding their unique preferences in interacting with erythrocytes from animals and humans, their sugar affinities, the critical amino acids for their functionality, the molecular weights of their subunits and their respective topologies, and ultimately, their dimerization and 3D β-prism-II structure, which reportedly diverge from those observed in other GNA-related lectins. These discrepancies not only deepen our understanding of monocot lectins but also render these proteins inherently captivating. This review marks the inaugural attempt at consolidating almost all published reports on lectins from the <em>Araceae</em> family, with the aim of furnishing glycobiology scientists with essential insights into potential laboratory challenges, the characteristics of these lectins, and avenues for future research.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141544506","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain","authors":"Chrislaine Withers-Martinez ,&nbsp;Roger George ,&nbsp;Sarah Maslen ,&nbsp;Létitia Jean ,&nbsp;Fiona Hackett ,&nbsp;Mark Skehel ,&nbsp;Michael J. Blackman","doi":"10.1016/j.bbagen.2024.130665","DOIUrl":"10.1016/j.bbagen.2024.130665","url":null,"abstract":"<div><h3>Background</h3><p>The malaria parasite <em>Plasmodium falciparum</em> replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31.</p></div><div><h3>Methods</h3><p>Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX.</p></div><div><h3>Results</h3><p>We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity.</p></div><div><h3>Conclusions</h3><p>Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity.</p></div><div><h3>General significance</h3><p>Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0304416524001089/pdfft?md5=7103e10c9071c61fc21e0d011e008abb&pid=1-s2.0-S0304416524001089-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"YAP1-mediated dysregulation of ACE-ACE2 activity augments cardiac fibrosis upon induction of hyperglycemic stress","authors":"Arunima Mondal ,&nbsp;Shreya Das ,&nbsp;Madhuchhanda Das ,&nbsp;Santanu Chakraborty ,&nbsp;Arunima Sengupta","doi":"10.1016/j.bbagen.2024.130666","DOIUrl":"10.1016/j.bbagen.2024.130666","url":null,"abstract":"<div><h3>Background</h3><p>Diabetic stress acts on the cardiac tissue to induce cardiac hypertrophy and fibrosis. Diabetes induced activated renin angiotensin system (RAS) has been reported to play a critical role in mediating cardiac hypertrophy and fibrosis. Angiotensin converting enzyme (ACE) in producing Angiotensin-II, promotes cardiomyocyte hypertrophy and fibrotic damage. ACE2, a recently discovered molecule structurally homologous to ACE, has been reported to be beneficial in reducing the effect of RAS driven pathologies.</p></div><div><h3>Methods</h3><p><em>In vivo</em> diabetic mouse model was used and co-labelling immunostaining assay have been performed to analyse the fibrotic remodeling and involvement of associated target signaling molecules in mouse heart tissue. For <em>in vitro</em> analyses, qPCR and western blot experiments were performed in different groups for RNA and protein expression analyses.</p></div><div><h3>Results</h3><p>Fibrosis markers were observed to be upregulated in the diabetic mouse heart tissue as well as in high glucose treated fibroblast and cardiomyocyte cells. Hyperglycemia induced overexpression of YAP1 leads to increased expression of β-catenin (CTNNB1) and ACE with downregulated ACE2 expression. The differential expression of ACE/ACE2 promotes TGFB1-SMAD2/3 pathway in the hyperglycemic cardiomyocyte and fibroblast resulting in increased cardiac fibrotic remodeling.</p></div><div><h3>Conclusion</h3><p>In the following study, we have reported YAP1 modulates the RAS signaling pathway by inducing ACE and inhibiting ACE2 activity to augment cardiomyocyte hypertrophy and fibrosis in hyperglycemic condition. Furthermore, we have shown that hyperglycemia induced dysregulation of ACE-ACE2 activity by YAP1 promotes cardiac fibrosis through β-catenin/TGFB1 dependent pathway.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141490711","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Chinese medaka (Oryzias sinensis) dmrt1 gene converts females to males in medaka (Oryzias latipes)","authors":"Lei Chen ,&nbsp;Yan Huang ,&nbsp;Qi-Hua Pan ,&nbsp;Meng-Yang Wang ,&nbsp;Jing-Jie Liang ,&nbsp;Tian-Sheng Chen","doi":"10.1016/j.bbagen.2024.130664","DOIUrl":"10.1016/j.bbagen.2024.130664","url":null,"abstract":"<div><h3>Background</h3><p>Chinese medaka (<em>Oryzias sinensis</em>) is widely distributed in freshwater rivers in China. Similar to the medaka (<em>Oryzias latipes</em>), Chinese medaka has the characteristics of small size, rapid reproductive cycle, and strong adaptability, which makes it suitable as a model organism for studies in basic biology and environmental toxicology. Chinese medaka exhibits distinct sexual dimorphism. However, due to the lack of complete genomic information, the regulation of sex determination and differentiation-related genes in Chinese medaka remains unclear.</p></div><div><h3>Methods</h3><p>Chinese medaka <em>dmrt1</em> (Os<em>dmrt1</em>) was cloned by PCR, and transgenic individuals of medaka [Tg(CMV:Os<em>dmrt1</em>)] overexpressing Os<em>dmrt1</em> were generated to investigate the role of Os<em>dmrt1</em> in sex determination. Western blot was used to validate the integration of the Os<em>dmrt1</em> into the medaka genome. Tissue sectioning and HE staining were used to identify Tg(CMV:Os<em>dmrt1</em>) physiological gender and phenotype. qRT-PCR was used to analyze the expression of gonad-specific genes.</p></div><div><h3>Results</h3><p>Os<em>dmrt1</em> was cloned and identified, and it shared similar evolutionary relationships with medaka <em>dmrt1</em>. Tg(CMV:Os<em>dmrt1</em>) exhibited partial sex reversal from female to male in the F2 generation, with genetically female individuals developing testes and producing functional sperm. Additionally, the secondary sexual characteristics of the transgenic females also changed to males.</p></div><div><h3>Conclusion</h3><p>The Chinese medaka <em>dmrt1</em> gene could convert females to males in medaka.</p><p><em><strong>General significance:</strong></em> These results not only elucidate the function of Chinese medaka <em>dmrt1</em>, but also accumulate knowledge for studying the function of economically important fish genes in model fish by transgenic technology.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141465913","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity","authors":"Yuta Hashimoto ,&nbsp;Haruka Kawade ,&nbsp;WanXue Bao ,&nbsp;Sayaka Morii ,&nbsp;Miyako Nakano ,&nbsp;Masamichi Nagae ,&nbsp;Reiko Murakami ,&nbsp;Yuko Tokoro ,&nbsp;Misaki Nakashima ,&nbsp;Zixuan Cai ,&nbsp;Tomoya Isaji ,&nbsp;Jianguo Gu ,&nbsp;Kazuki Nakajima ,&nbsp;Yasuhiko Kizuka","doi":"10.1016/j.bbagen.2024.130663","DOIUrl":"10.1016/j.bbagen.2024.130663","url":null,"abstract":"<div><h3>Background</h3><p><em>N</em>-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific <em>N</em>-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses <em>N</em>-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells.</p></div><div><h3>Methods</h3><p>Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed <em>in vitro</em> activity assay, lectin blotting analysis and <em>N</em>-glycomic analysis using point mutants to assess their activity.</p></div><div><h3>Results</h3><p>Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower <em>in vitro</em> activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the <em>cis</em> side of the Golgi, probably allowing for prior action to competing galactosyltransferases.</p></div><div><h3>Conclusions</h3><p>The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity.</p></div><div><h3>General significance</h3><p>Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.</p></div>","PeriodicalId":8800,"journal":{"name":"Biochimica et biophysica acta. General subjects","volume":null,"pages":null},"PeriodicalIF":2.8,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141465914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}