bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross‐talks between intracellular zinc increases and reactive oxygen species in hypoxia
Authors: Slepchenko Kira , Li Yang V 
Published: November 2023

Zinc recently has emerged as a contributing factor in cellular injury and death caused by hypoxia. Reactive oxygen species (ROS) are also known to accumulate during hypoxia, and are damaging to normal cellular function. We hypothesized that there is a connection between these two important signaling events with potential cause and effect relationship between them. To test the hypothesis, we used fluorescent probes to visualize both free cytosolic zinc (FluoZin‐3, AM) and mitochondrial ROS (MitoSOX Red) in live HeLa cells undergoing hypoxia. We observed a sharp increase in free cytosolic zinc within 60 seconds of hypoxia, which was followed by rapid decrease in free zinc. This zinc response resembled a wave because it had the ascending and descending component, we named it phase I (ascending) and phase II (descending). There was a latency period observed, were the zinc levels remained elevated compared to baseline but did not change during this phase. The latency period is a III phase of the hypoxic zinc response. There is a second significant zinc increase after 18–20 minutes of hypoxia and we named it IV phase of zinc response. This is the first detailed description of the initial zinc transients during hypoxia, where we describe four distinct phases. The first mitochondrial ROS increase started after 5 minutes of hypoxic exposure, then leveled off. These data show that zinc increase preceded the initial ROS increase. Removal of intracellular zinc with zinc chelator TPEN during hypoxia caused mitochondrial ROS accumulation to be delayed about 10 minutes, showing that when zinc is removed, the initial mitochondrial ROS accumulation is not initiated, suggesting that zinc plays a major role in initiating mitochondrial ROS increase. In normoxic conditions, the application of exogenous zinc by itself induced mitochondrial ROS accumulation, adding to the evidence that zinc plays a key role in initiating mitochondrial ROS increase. We hypothesized that NADPH oxidase, a known ROS producer, may be a link between zinc and mitochondrial ROS. In cells that were exposed to exogenous zinc and NADPH oxidase was inhibited by apocynin, mitochondrial ROS was not observed, confirming that NADPH oxidase plays an important role in zinc induced ROS increase. Western blot of total extracts of HeLa cells subjected to exogenous zinc showed the higher level of NADPH oxidase when compared to cells without zinc treatment. Thus, these results suggest a new mechanism of cross‐talk between zinc and mitochondrial ROS, where free cytosolic zinc increase precedes and causes initial mitochondrial ROS increase with zinc and NADPH oxidase as key players. The first ROS increase injures mitochondria and they in turn release ROS and zinc, further contributing to cell injury. Hypoxia is an underlying cause of many important diseases like stroke, heart attack and pulmonary embolism and these findings further our understanding of cellular response to hypoxia.Support or Funding InformationNIH (NS081629 to Y.V. Li)
A conserved putative kinase is required for coenzyme Q biosynthesis: Functional insights from yeast genetics
Authors: Pham Nguyen Vo Bao , Clarke Catherine 
Published: November 2023

Coenzyme Q (ubiquinone or Q) is an essential lipid functioning as electron carrier in the electron transport chain during cellular respiration. At least 11 Coq polypeptides involved in the Q biosynthetic pathway have been characterized in Saccharomyces cerevisiae. Several of the Coq polypeptides essential for efficient Q biosynthesis organize into a high weight molecular complex localized to the inner mitochondrial membrane, designated the ‘CoQ‐synthome’. Coq8 was identified as an atypical kinase responsible for sustaining the stability of the CoQ synthome. Overexpression of COQ8 has been shown to restore steady state levels of Coq proteins in several of the yeast coq null mutants. However, the specific function of Coq8 remains unknown. Previously, a mutant yeast strain harboring the coq8‐3 allele was shown to encode the A197W substitution within the conserved kinase motif 1, and was suspected to disrupt the putative kinase activity of Coq8p. The yeast coq8‐3 mutant lacks Q, is respiratory deficient, and does not grow on non‐fermentable media. The yeast coq8‐3 mutant exhibits a dramatic decrease in the steady‐state levels of the Coq4 and Coq9 polypeptides. To gain insight into Coq8 function, the coq8‐3 mutant was selected for further investigation. Six spontaneous revertants were isolated from the coq8‐3 mutant. Each of the six revertants was able to grow on a non‐fermentable carbon source and retained the parental coq8‐3 point mutation. Analysis of the six revertants by LC/MS‐MS revealed Q6 content was restored. Three out of six revertants appeared to contain recessive secondary mutations, while one revertant displayed a dominant supressor mutation as demonstrated by complementation tests. Here we report our investigation of the spontaneous revertants with recessive suppressor mutations. Elucidation of how they function to restore Q biosynthesis in the coq8‐3 mutant may help clarify the function of Coq8 in Q biosynthesis. This research was supported by NSF MCB‐1330803.Support or Funding InformationThis research was supported by NSF MCB‐1330803.
Four Acyltransferases Uniquely Contribute to Phospholipid Heterogeneity in Saccharomyces cerevisiae
Authors: Oelkers Peter , Pokhrel Keshav 
Published: November 2023

Diverse acyl‐CoA species and acyltransferase isoenzymes are part of a complex system that synthesizes glycerophospholipids and triacylglycerols. Saccharomyces cerevisiae, having four main acyl‐CoA species, two main glycerol‐3‐phosphate 1‐O‐acyltransferases (Gat1p, Gat2p) and two main 1‐acylglycerol‐3‐phosphate O‐acyltransferases (Lpt1p, Slc1p), provides an experimental system of reduced complexity. To comprehensively examine the in vivo contribution of the acyltransferase isoenzymes to phospholipid heterogeneity, haploid yeast with compound mutations were generated: gat1Δlpt1Δ, gat2Δlpt1Δ, gat1Δslc1Δ, and gat2Δslc1Δ. All mutations mildly reduced [3H]palmitic acid incorporation into phospholipids relative to triacylglycerol. Electrospray ionization tandem mass spectrometry analysis of phospholipids identified few differences from wild type in gat1Δlpt1Δ, dramatic differences in gat2Δslc1Δ, and intermediate changes in the other two mutants. The gat2Δslc1Δ yeast, solely expressing Gat1p and Lpt1p, had phospholipids with 10% more unsaturated acyl chains and 20% fewer species with 34 acyl chain carbons. These percent changes varied among phospholipid head group species. Thus, the combined activities of Gat2p and Slc1p seem to promote the production of phospholipids with “mixed” chain lengths (e.g. 16 and 18 carbons). This is particularly true for phosphatidylserine. Head group allocation was also affected in gat2Δslc1Δ yeast which had 15% more phosphatidylethanolamine, 38% less phosphatidylserine, and 85% less phosphatidylglycerol. These alterations slowed growth in rich media at 30 °C by 18% but not in media with non‐fermentable carbon sources. Growth was abrogated in rich media at 18.5 °C or containing 10% ethanol. Therefore, Gat2p and Slc1p primarily dictate appropriate phospholipid acyl chain composition, presumably through de novo synthesis, in several growth conditions. This composition is likely steered by Slc1p selectively pairing acyl chains of different lengths.Support or Funding InformationUniversity of Michigan‐Dearborn Office of Research and Sponsored Programs
Biochemical Characterization of a Mycobacterial Acyltransferase Involved in the Metabolism of Long‐Chain Fatty Acids
Authors: Law Jamison , Daniel Jaiyanth 
Published: November 2023

Latent tuberculosis affects about two billion people and is caused by dormant Mycobacterium tuberculosis (Mtb). In its dormant state, Mtb becomes phenotypically tolerant to antibiotics and accumulates triacylglycerol synthesized using fatty acids derived from host lipids. The Mtb acyltransferase catalyzing the first step of the triacylglycerol biosynthetic pathway has not been studied. Therefore, we cloned the open reading frame of a putative mycobacterial glycerol‐3‐phosphate acyltransferase (mGPAT) and expressed the protein in Escherichia coli to study its function. We observed that cell lysates expressing mGPAT synthesized more 14C‐labeled lysophosphatidic acid derived from 14C‐labeled glycerol‐3‐phosphate and palmitoyl‐CoA than lysates lacking the gene. This indicates that mGPAT is a functional gene in synthesizing the first product of the glycerophospholipid/triacylglycerol biosynthetic pathway. The cell lysates expressing mGPAT also displayed increased radiolabel accumulation in polar lipids suggesting that the product of mGPAT was utilized in lipid biosynthesis. E. coli cells expressing mGPAT exhibit higher growth rate during exponential phase compared to E. coli lacking mGPAT suggesting that mGPAT enhances the ability of E. coli to utilize long‐chain fatty acids for membrane lipid biosynthesis. Metabolic incorporation of 14C‐palmitate by intact E. coli cells into lipids was observed to be increased during early log phase when mGPAT was expressed. However, the metabolic incorporation of 14C‐acetate into lipids during exponential phase was decreased in cells expressing mGPAT. Our results suggest that mGPAT preferentially utilizes exogenous fatty acids for lipid biosynthesis over endogenously synthesized fatty acids and potentially inhibits lipid accumulation from fatty acids synthesized within the cell during the exponential phase. Ongoing studies seek to investigate the effect of mGPAT on lipid biosynthesis when expressed heterologously in Mycobacterium smegmatis.Support or Funding InformationDepartment of Biology, Indiana University‐Purdue University Fort Wayne; Purdue Research Foundation
XBP‐1: A Key Transcriptional Regulator of Lipid Synthesis in Renal Epithelial Cells
Authors: del Carmen Fernandez Maria , Malvicini Ricardo , Weber Karen , Parra Leandro , Lepera Leandro , Casali Cecilia 
Published: November 2023

Renal medullary collecting duct cells are physiologically exposed to high and variable concentrations of NaCl involved in urinary concentrating mechanism. Despite such adverse environment, renal cells activate protective and survival mechanisms. In these sense, our lab demonstrated that hyperosmolarity increased lipid biosynthesis to protect membrane integrity. It is also known that the transcription of osmoprotective genes such as organic osmolyte and urea transporters, COX2, and AQPs, are induced. Such a massive protein synthesis could cause endoplasmic reticulum (ER) stress and trigger unfolded‐protein response (UPR) signalling pathways, among them the Ire1‐ XBP1 pathway. The active form of XBP‐1 is a transcription factor that activates the expression of lipogenic genes which, in turn, activate membrane biogenesis and ER stress alleviation. Thus, it could exist a relationship between osmotic‐increased lipid metabolism and UPR activation. In the present work, we explore such a possibility. To do that, MDCK cells were subjected to iso (298 mOsm/kg H2O) or hyper osmolarity (512 mOsm/kg H2O) for different periods of time and then, lipid synthesis, UPR activation, and the relationship between both processes were studied. 24 and 48 h of hypertonic treatment significantly increased the levels of 14C‐glycerol labelled‐phospholipids (PLs) and triglycerides (TGs). Such increase was consistent with the increased mRNA levels of Lipin2, FAS, DGAT2 and SCD. ER stress markers mRNA XBP1 and CHOP were also increased. XBP1 silencing reduced the levels 1,2 DAG and TAG formed. This finding was consistent with the decrease in the levels of Lipin2 and DGAT2 mRNA. Interestingly, neither PL synthesis nor Kennedy pathway enzymes mRNA levels were affected. We also found that XBP1 silencing causes SREBP (a master lipogenic transcription factor) downregulation. These results clearly evidence a major role of XBP1 in the regulation of lipid synthesis in renal epithelial cells and cell protection against osmotic stress.Support or Funding InformationFONCyT. Prestamo BID‐PICT2013‐1132.
Genetic dissection of early endosomal recycling highlights a TORC1‐independent role for Rag GTPases
Authors: MacDonald Chris , Piper Robert C 
Published: November 2023

Endocytosed cell surface membrane proteins rely on various recycling pathways for their return to the plasma membrane1. Although endosome‐to‐plasma membrane recycling is critical for many cellular processes2, much of the required machinery is unknown. We discovered that yeast also has a recycling route to the cell surface that bypasses the Golgi. Using an engineered synthetic reporter that exclusively follows this pathway, we performed a genetic screen to reveal a cohort of novel and conserved regulators of direct endosome to cell surface recycling. One critical component was the Rag GTPases, Gtr1 and Gtr2, which worked downstream of the exchange factor Vam6 and upstream of the Gtr1 effector Ltv1. Although the Rag GTPases are known to activate TORC1 on the vacuole/lysosome3–5, we find the Vam6>Gtr1‐Gtr2>Ltv1 recycling pathway is independent of TORC1, thus establishing a distinct role for these GTPases in early endosome recycling.Support or Funding InformationRO1GM058202
Structural and functional studies of the ANTH domain as ubiquitin –binding module involved in endocytosis
Authors: Pashkova Natalja , Piper Robert C. 
Published: November 2023

Ubiquitin serves as a sorting signal throughout the endocytic pathway by being recognized by a series of ubiquitin‐sorting receptors. Ubiquitin modification also serves as a means to regulate the assembly and function of the machinery that executes endocytosis. Using a newly developed large‐scale computational‐based yeast 2‐hybrid approach, we identified a key the AP180 N‐ terminal homology domain ANTH) as a novel ubiquitin binding module. ANTH domains are distributed in a variety of proteins such as AP180, CALM, HIP1, and HIP1R implicated in providing adaptor function for clathrin‐mediated endocytosis. We find ANTH domains from a variety of proteins bound mono‐ubiquitin with relatively high affinity and showed preference for K63 linked polyubiquitin over K48‐linked polyubiquitin. We solved the crystal structure of CALM ANTH domain in a complex with ubiquitin at 2.8 A. The ubiquitin binding site resides at C‐terminal part of the ANTH domain whereas PtdIns(4,5)P2 binding site is at its N‐terminus. Ubiquitin also occupies an overlapping surface that is partly used to bind SNARE proteins such as Vamp8 and Vamp7. Paramagnetic relaxation enhancement NMR experiments independently verified the orientation of the bound ubiquitin, which guided engineering of point mutations that abolished ubiquitin‐binding while preserving binding to SNARE proteins and PtdIns(4,5)P2 mutations in ubiquitin‐binding interface of CALM ANTH protein completely abolished ubiquitin binding. CALM and AP180 proteins function as adaptors in sorting of R‐SNARE proteins via interaction of ANTH domain with SNARE motifs. We found that the ubiquitin binding mutant CALM ANTH domain is still capable of binding to the SNARE protein Vamp 7. Moreover, structure guided mutagenesis allowed isolation of similar mutants within the yeast ANTH proteins Sla2, YAP1801, and YAP1802 that were specifically defective in ubiquitin‐binding. Yeast strains carrying these mutant alleles showed altered trafficking of the GPCR Ste2, which uses a ubiquitin‐dependent pathway for internalization.Our study suggest that ubiquitin‐binding to ANTH domains may provide a means to recognize ubiquitinated cargo and incorporate them into clathrin‐coated vesicles for internalization.Support or Funding InformationNIH: 5R01GM058202
Glycosylation quality control by the Golgi structure
Authors: Wang Yanzhuang , Huang Shijiao , Haga Yoshimi , Kweon Hye Kyong Kyong , HIRAYAMA Hiroto , Andrews Philip , Suzuki Tadashi 
Published: November 2023

Glycosylation is a ubiquitous and essential modification that occurs on proteins and lipids in all living cells. Asparagine‐linked protein N‐glycosylation, the most complex glycosylation, initiates in the endoplasmic reticulum (ER) and matures in the Golgi apparatus. This process not only requires an accurate distribution of processing machineries, such as glycosyltransferases, glycosidases and nucleotide sugar transporters, but also needs an efficient, and well‐organized factory that is responsible for the fidelity and quality control of sugar chain processing. In addition, accurate glycosylation must occur in coordination with protein trafficking and sorting. These activities are carried out by the Golgi apparatus, a membrane organelle in the center of the secretory pathway. To accomplish these tasks, the Golgi has developed into a unique stacked structure of closely aligned flattened cisternae in which Golgi enzymes reside. We have developed a multidisciplinary approach to dissect the molecular mechanism of Golgi structure formation and its significance in protein glycosylation [1]. We found that Golgi cisternal stacking is required for accurate glycosylation in interphase cells [2,3]. We also found that in mitosis, key Golgi enzymes are inactivated This work has significant impact in understanding human diseases in which the Golgi becomes abnormal. For example, in Alzheimer's disease the Golgi is abnormally fragmented, which accelerate amyloidocleavage of the amyloid beta (Aβ) precursor protein APP. Significantly, rescue of the Golgi structure reduces Aβ production [4]. Our study suggests the Golgi as a potential drug target for disease treatment. In addition, we have made surprising discoveries that some Golgi proteins are O‐GlcNAcylated and some Golgi glycosylation enzymes are regulated by phosphorylation during the cell cycle.Support or Funding InformationNIH R01s NIH GM087364 and GM112786.
Towards a glycosidase toolbox for glycan structure analysis: General Label‐Free Mass Spectrometry‐Based Assay to Identify Glycosidase Substrate Competence
Authors: Peng Tianyuan , Nagy Gabe , Pohl Nicola L. B. 
Published: November 2023

Glycosidases are enzymes that catalyze the hydrolysis of glycosyl linkages and are widely used in oligosaccharide linkage analysis1. Common glycosidase assays, however, rely on using derivatized non‐natural substrates for analysis. Information as to the specificity of the leaving group and kinetic competency among natural substrates are lost during the process. Detection and quantitation of carbohydrate analytes without derivatization remains difficult to achieve. Herein we report two putative thermophilic glycosidase genes that were codon optimized and expressed, a mass‐differentiated substrate library2 to screen and identify the chemical function of the newly expressed glycosidases, and a novel mass spectrometric glycosidase assay developed to couple both the enzymatic hydrolysis of glycosidic linkages using newly characterized thermophilic glycosidases with the detection and quantitation of D‐galactose, D‐mannose and D‐glucose using a fixed ligand kinetic method3,4. Various natural substrates were screened using this new label‐free assay5 and kinetic competency information was first reported for two thermophilic enzymes. These newly characterized glycosidases were then combined with commercial glycosidases to form a glycosidase toolbox that enables a fast enzymatic screening for linkage analysis of glycan samples using a high throughput experiment platform. Overall, this assay is both fast and sensitive in that it only takes a few hours to detect nanogram quantities of monosaccharides and can quickly provide a complete cleavage profile for a glycosidase or oligosaccharide of interest.Support or Funding InformationWe appreciate the funding support of Joan and Marvin Carmack fund
Analysis of C‐Mannosylated Proteins, Hsc70, and Inflammation
Authors: Seccurro Drake Anthony , Rakus John 
Published: November 2023

C‐mannosylation is a unique type of post‐translational modification composed of a mannose attached to the indole functional group, most commonly to the amino acid sequence Trp‐xaa‐xaa‐Trp through a carbon‐carbon bond. Many of the functions of C‐linked glycosylation have yet to be defined. Some proteins with the C‐mannosylation composition have been found to be key factors in the process of inflammation (Tuoen et al., 2012). Heat Shock Cognate protein 70 (HSC70) is a molecular chaperone that is constitutively expressed, and has been classified as having a high affinity for C‐mannosylated peptides (Ihara et al., 2010). While HSC70 is known to regulate various cellular functions, its interactions with many other molecules remains unknown. However, HSC70 has been discovered to have a vital role in the myocardial inflammatory response, especially after ischemia and reperfusion injury. (Ning et al., 2008). In a study conducted by Amgen Incorporated, in vitro models were designed to examine the effects of ischemia on myocytes using coverslip hypoxia. Myocytes cultured in these conditions showed metabolic activity that caused inflammation and necrosis, but more importantly showed an effective model of ischemic conditions (Pitts et al., 2004).The aim of this experiment is to use similar methods to induce hypoxic conditions to stimulate the upregulation of HSC70 in RAW264.7 cells. By upregulating and isolating HSC70 in ischemic conditions, there is possibility of extracting C‐mannosylated glycoproteins associated with the protein. We use immunoprecipitation of HSC70 to identify ligands that are upregulated in ischemic conditions which are sequenced by tandem mass spectrometry.Support or Funding Information
The Tumor Associated Lectin Galectin‐3 Can Bind and Sequester Cancer Biomarkers
Authors: Fan Ni , Talaga Melanie , Brown Robert , Fueri Ashli , Bandyopadhyay Purnima , Dam Tarun 
Published: November 2023

Most covalently‐linked glycans of glycoprotein cancer biomarkers are potential binding epitopes of tumor‐associated endogenous lectin galectin‐3 (Gal‐3). As glycoprotein biomarkers and Gal‐3 could be secreted by the same tumor cells, they could exist in the same vicinity. We hypothesized that Gal‐3 and the respective biomarker could engage in high affinity binding due to their close proximity. Such binding could result in mutual sequestration through cross‐linked complex formation. We tested our hypothesis by using Thyroglobulin (Tg) and human Gal‐3. Tg, a thyroidal glycoprotein, is a pre‐ and postoperative biomarker of differentiated thyroid cancer (DTC) and Gal‐3 is secreted by malignant thyroid cells. We found that these two biomarkers, produced by thyroid cancer cells, interacted with each other with submicromolar affinity and formed insoluble cross‐linked complexes at their stoichiometric concentrations. One Tg molecule bound up to 14 molecules of Gal‐3. Such complex formation mutually locked both Tg and Gal‐3 in cross‐linked complexes, decreasing the concentration of their freely available forms in the solutions. Formation of the Tg–Gal‐3 complexes was reversible as preformed complexes were found to be sensitive to free Tg and free Gal‐3. Both free Tg and Gal‐3 individually dissolved preformed Tg–Gal‐3 complexes in a concentration‐dependent manner. Complex formation between Tg and Gal‐3 and the sensitivity of the complexes to free Tg and Gal‐3 can potentially influence their biological functions. Sequestration of Tg by Gal‐3 in cross‐linked complexes might also interfere with their clinical detection, the same way Tg autoantibody (TgAb) is reported to interfere in clinical Tg assays. The data presented here also support a model of Gal‐3‐mediated homeostasis of Tg. The present findings could be relevant to other cancers that produce glycoprotein biomarkers and Gal‐3 [1].Support or Funding InformationMichigan Technological University