bluuebunny/crossref_metadata_2025 huggingface dataset:
S‐nitrosylation Is Responsible for Muscle Atrophy and Weakness in GNE Myopathy
Authors: Miyakawa Miho , Cho Anna , Malicdan May Christine V , Nishino Ichizo , Noguchi Satoru
Published: November 2023

Background

Sialic acids are the most abundant terminal monosaccharides on glycoconjugates of eukaryotic cell surfaces and are involved in a variety of cellular functions. UDP‐N‐acetylglucosamine‐2‐epimerase/N‐acetylmannosamine 6‐kinase is a rate‐limiting enzyme for sialic acid biosynthesis, which is encoded by GNE gene. Mutations of GNE causes GNE myopathy, which is debilitating autosomal recessive myopathy, and is clinically characterized by atrophy and weakness in distal skeletal muscles. Previously, we have evidenced that sialic acid deficiency plays a key role for the pathogenesis of this myopathy with the GNE myopathy model mice, presenting muscle atrophy and weakness followed by myofiber degeneration. The phenotype was attenuated by sialic acid supplementation. And also, we found the elevation of reactive oxygen species in skeletal muscles of the model mice and demonstrated a therapeutic effect of an anti‐oxidant, N‐acetylcysteine (NAC) on muscle atrophy as well.

Objective

We aimed to identify protein modification(s) produced by ROS, which cause muscle atrophy and weakness in GNE myopathy.

Methods

For the analysis on S‐nitrosylation status using frozen skeletal muscles, we switched S‐nitrosocysteines to biotin‐labeled cysteins and purified the S‐nirosylated proteins by Neutravidin beads, followed by LC‐tandem mass shotgun analyses or western blotting.

Results

We found that protein S‐nitrosylation was increased in GNE myopathy muscles. By proteome analysis, more than 60 proteins were identified in the S‐nitrosylated group, which revealed that the major proteins were involved in muscle contraction (actin and myosins) and energy production (muscle creatin kinase, enolase 3 and aldolase). We also found that sialic acid supplement significantly decreased S‐nitrosylation of these proteins as much as NAC treatment did.

Discussion

Our results suggest that hyposialylation leads to failure in ROS scavenging and promotes S‐nitroso modification to deteriorate muscle contraction. Therefore, ROS cancelling would be a promising strategy to prevent muscle weakness and atrophy as well as to cure GNE myopathy.

Support or Funding Information

This study was partially supported by Intramural Research Grant (28‐6) for Neurological and Psychiatric Disorders of NCNP, by Comprehensive Research on Disability Health and Welfare (H25‐Shinkei Kin‐Ippan‐004) from Japan Agency for Medical Research and Development, AMED and by JSPS KAKENHI (23390236).
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 Information

NIH (NS081629 to Y.V. Li)
Mitochondria‐targeted catalase primes adipocyte differentiation through regulation of Pref‐1
Authors: Tavares Kendall , Christian Brooke
Published: November 2023

Obesity is caused by either an increase in the number of fat cells, an increase in the size of fat cells, or both. Fat cells increase in number when adipocyte precursor cells differentiate into mature fat cells, a process previously acknowledged as dependent on the presence of reactive oxygen species. Antioxidants decrease the amount of reactive oxygen species and reduce adipocyte differentiation. Despite this, a preliminary study found that mitochondria‐targeted expression of the antioxidant catalase caused adipocyte precursors to initially differentiate more quickly than corresponding cells from wild type mice. This increase in differentiation occurred in the first 5 days but was reversed by day 7. Preliminary qRT‐PCR data indicates that cells with mitochondria‐targeted catalase differentiate faster due to the absence of the transcriptional downregulator Pref‐1. The downregulation of Pref‐1 stimulates expression of genes that promote differentiation such as the master regulator PPARγ2 and targets Perilipin, aP2, FASN and AdipoQ. The relationship between catalase and Pref‐1 is currently under investigation. Results from this study may have implications on the potential use of antioxidants as treatments for obesity.
Lack of Efficient Metabolism Adaption Caused Failure of Regenerative Cell‐Based Therapy in a Rat Model of Metabolic Syndrome
Authors: Jamaiyar Anurag , Wan Weiguo , Janota Danielle , Enrick Molly , Ohanyan Vahagn , Yin Liya , Chilian William
Published: November 2023

Nearly 30–40% of patients with coronary disease show little or no coronary collateral growth and have a poorer prognosis than those with well‐developed collaterals. Stem cell therapies hold promise for stimulating vascular growth in the heart, but most results to date have been modest. Perhaps, the lack of efficient metabolic adaption of stem cells from optimized cell culture medium to hostile environment such as metabolic syndrome in patients caused the compromised stem cell function. To test this, we determined if cell‐based regenerative therapies stimulate coronary collateral growth in a rat model characterized by oxidative stress and poor coronary collateralization. Previously we found that induced vascular progenitor cells (iVPCs) were the best cell type (compared to MSCs and iPS cells) to stimulate coronary collateral growth in lean (LN) rats (10‐day repetitive ischemia [RI] protocol). Using the RI protocol, iVPCs were implanted intramuscularly in the heart of Zucker Obese Fatty (ZOF) rats. Coronary collateral flow was significantly increased by iVPC in LN but not in ZOF suggesting that with the metabolic syndrome iVPCs fail to induce coronary collateral growth. To understand why iVPCs did not stimulate collateral growth in ZOF rats, we performed mechanistic studies and investigated the relationship of the stemness and function of the stem cells to the metabolic environment. We cultured the partially reprogrammed cells in the medium with Zucker Lean and Zucker Obese serum or with different concentration of glucose and studied the cellular metabolic phenotype and bioenergetics by Seahorse XF24 Analyzer and cell lineage marker change and cell differentiation capability by real‐time PCR and FACS. We found that stem cell metabolic phenotype switched under the condition of oxidative stress and high glucose, which is consistent with the change of stem cell function and differentiation capability under the condition of oxidative stress and high glucose. We hypothesize that mitochondrial metabolic potential is key to the adaption process. Therefore, our study suggested that efficacy of partially reprogrammed VSMCs or iVPCs decreased because of the lack of efficient metabolic adaption from optimized cell culture medium with all growth factors and nutrient supplements to hostile environment in Zucker Obese rat with hyperlipidemia, high glucose and insulin resistance.
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 Information

This 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 Information

University 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 Information

Department of Biology, Indiana University‐Purdue University Fort Wayne; Purdue Research Foundation
Insights into the cellular trafficking of perilipin 5
Authors: Bailey Hannah M. , Tansey John T.
Published: November 2023

Perilipins are a family of proteins found on lipid storage droplets that play a role in the regulation and metabolism of cellular neutral lipid. Perilipin 5 has also been found localized elsewhere in the cell including the cytoplasm, mitochondria, endoplasmic reticulum, and more recently, the nucleus. The localization of perilipin 5 in these locations is not random, but regulated in part through phosphorylation of perilipin 5. We hypothesized that the movement of perilipin 5 is regulated through interactions with trafficking proteins. This work seeks to identify the mechanisms and interaction partners involved in phosphorylation dependent perilipin 5 movement in the cell. Following stimulation of the PKA signaling pathway with forskolin and isobutylmethyl xanthine, we observed perilipin 5 localization in the nucleus. The movement of perilipin 5 to the nucleus is time dependent taking minutes to hours to occur. We have identified three weak putative, nuclear localization sequences in the perilipin 5 sequence. Based on these observations, interaction partner(s) must aid the movement of the protein in the cell and transit into the nucleus. These partners may include chaperones, Rabs, importins, or other trafficking proteins. Currently, we are investigating potential interactions between Perilipin 5 and Rabs 32 and 19 using immunofluorescent coverslips and western blotting. Collectively these data indicate that the trafficking of perilipin 5 plays an important role in the function of this protein in the cell.
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 Information

FONCyT. 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 Information

RO1GM058202
Cytoskeleton‐Associated Protein 4 (CKAP4) Knockdown Disrupts the Actin Filament Network in Bladder Carcinoma Cells
Authors: Majernick Thomas , Chavda Burzin , Planey Sonia Lobo
Published: November 2023

Cytoskeleton‐associated protein 4 (CKAP4) is a 63kDa, non‐glycosylated type II integral membrane protein that plays an essential role in the maintenance of endoplasmic reticulum structure by anchoring the rough ER to microtubules in epithelial cells. More recently, CKAP4 has been identified as a biologically relevant receptor for four unique ligands—tissue plasminogen activator, surfactant protein A, Dickkopf1, and antiproliferative factor. While research shows that CKAP4 interacts directly with the microtubule network, its potential role within the actin filament network has not been previously studied. To explore this putative role, we used RNA interference to reduce CKAP4 expression in T24 bladder carcinoma cells and then fixed and stained the actin cytoskeleton using phalloidin‐488. We observed a marked difference in actin filament morphology and a disruption of the actin filament network in cells treated with CKAP4 siRNA versus cells treated with control siRNA or vehicle alone; moreover, CKAP4 knock down generated a significant reduction in actin polymerization. To understand the observed phenotypic changes, we performed differential gene expression analysis using quantitative PCR. Our data showed that CKAP4 knock down led to an increase in the expression of genes known to promote cell motility and dynamic changes in the cytoskeleton with a concomitant reduction in the expression of genes involved in stress fiber formation. Western blot analysis of selected, differentially expressed proteins supported the gene expression data. Collectively, these results suggest that CKAP4 may play an important role in regulating the actin cytoskeleton.
Investigating the Effects of Vps45 Mutations Associated with Severe Congenital Neutropenia on SNARE Interactions
Authors: D'Ordine Alexandra , Newburger Peter E , Munson Mary
Published: November 2023

In order to grow and survive, eukaryotic cells must accurately and efficiently transport cargo molecules into, out of, and between the different organelles in the cell. One essential mode of trafficking uses vesicles, membrane‐enclosed compartments. Several classes of proteins are involved in vesicle trafficking, including soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18‐like (SM) proteins. To promote fusion by bringing the vesicle and target membranes together, individual SNAREs form a SNARE complex, a process controlled by SM proteins. Vps45 is an SM protein that regulates SNARE complex formation in the endosomal pathway. In yeast, Vps45 has been shown to interact with its binding partner SNARE, the yeast homolog of mammalian syntaxin 16, Tlg2, to control its entry into the SNARE complex. By regulating SNARE complex assembly, Vps45 ensures that vesicle fusion in the endosomal pathway occurs in a controlled manner. Recently, several studies identified three independent single amino acid substitutions associated with the immune disorder severe congenital neutropenia, which is characterized by low levels of neutrophils and susceptibility to infection. This is caused by incomplete differentiation of neutrophils and the tendency of neutrophils that do develop to undergo apoptosis. However, it is unclear how these mutations lead to the disease. This study uses in vitro binding assays to investigate differences in affinities between the Vps45 mutants and the SNAREs. These results could suggest a molecular mechanism by which these mutations contribute to low neutrophil counts in patients with severe congenital neutropenia.
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 Information

NIH: 5R01GM058202
The selective autophagy pathway of nanodiamond‐SSEA‐1 antibody in GBM cells
Authors: Ting‐Hua Chen
Published: November 2023

Nanodiamond (ND) is a biocompatible carbon‐based nanomaterial that has been developed for cellular labeling and detection. Previously, we found that Ub (ubiquitin)‐coated nanodiamonds bind to autophagy receptors, including SQSTM1 (sequestosome 1), OPTN (optineurin), and CALCOCO2/NDP52 (calcium binding and coiled‐coil domain 2) and the LC3 (microtubule‐associated protein 1 light chain 3) for entry into the selective autophagy pathway, to deliver the cargos to lysosomes. In this study, we further reported that ND conjugated to a specific SSEA‐1 antibody (Ab) that can specifically bind to SSEA‐1, which is the marker of glioblastoma multiforme stem cells, and enter the selective autophagy pathway. ND‐SSEA‐1 Ab could bind to the SSEA‐1 proteins and increased the uptake ability of NDs in S1R1 (SSEA‐1 positive) cells but not in the GBM8901 (SSEA‐1 negative) cells. The ND‐SSEA‐1 Ab could bind with SSEA‐1 proteins by protein G beads using immunoprecipitation assays and observed under confocal microscope. Interestingly, the NDs were coated with Ub and then recognized by autophagy receptors to transport to the lysosomes in the S1R1 cells after treatment with ND‐SSEA‐1. We suggest that ND‐SSEA‐1 can specifically recognize the SSEA‐1 proteins on the GBM cells and delivered to lysosome through the selective autophagy pathway. These findings provide that the delivery pathway of antibody‐conjugated nanoparticles, which may develop biomedical applications including cancer stem cell labeling and tracking.
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 Information

NIH 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 Information

We 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 Information

Michigan Technological University
Impact of Provitamin A Biofortified Maize Consumption on Carotenoid Status of Zambian Children
Authors: Craft Neal E , Palmer Amanda , Schulze Kerry , Chileshe Justin , Barffour Maxwell , Siamusantu Ward , Klemm Rolf , West Keith
Published: November 2023

Objectives

Provitamin A carotenoid biofortified “orange” maize has been developed as a vitamin A deficiency control strategy using selective breeding for the β‐carotene hydroxylase‐1 gene, which increases β‐carotene, β‐cryptoxanthin, and zeaxanthin in the endosperm. We conducted a cluster‐randomized trial to test the impact of orange maize consumption (a predominantly β‐carotene‐rich variety) on serum retinol concentration in rural Zambian children (4–8 y). In the present analysis, we tested the intervention's impact on circulating carotenoids.

Methods

Children (n=1024, 4–8 y) were cluster‐randomized to receive 200 g dry weight/d orange or conventional “white” maize prepared with standardized low‐vitamin A relishes, 6 d/w for 6 m. The carotenoid profile was assessed in baseline and follow‐up serum samples in a subset (n=321 white, n=358 orange) of children. We assessed differences in log‐transformed carotenoids, expressed as geometric means and 95% confidence limits, accounting for cluster randomization.

Results

Baseline carotenoids did not differ between groups. There were no differences in lycopene and lutein between groups at follow‐up. Other carotenoids increased significantly (p<0.001) in orange vs white groups following the six‐month intervention: β‐carotene [273 (254,292) vs 147 (135,160) nmol/L], α‐carotene [19.7 (18.4,21.1) vs 10.6 (9.9,11.3) nmol/L], β‐cryptoxanthin [36.1 (33.6,38.8) vs 12.6 (11.8,13.5) nmol/L], α‐cryptoxanthin [18.7 (17.4,20.1) vs 8.9 (8.4,9.4) nmol/L], zeaxanthin [127 (119,135) vs 81 (75,88) nmol/L].

Conclusions

Children regularly consuming a biofortified maize variety bred primarily for β‐carotene had higher concentrations of other provitamin A carotenoids and zeaxanthin, suggesting the potential for health benefits beyond vitamin A deficiency control.

Support or Funding Information

Supported by HarvestPlus.
Pregestational obesity may have an indirect effect on iron status during pregnancy considering the increase of gestational weight
Authors: Flores‐Quijano Maria Eugenia , López‐Alarcón Mardya , Muñoz‐Manrique Cinthya , Tolentino‐Dolores Maricruz
Published: November 2023

Objective

To evaluate the influence of maternal pre pregnancy obesity on iron status during pregnancy

Methods

A longitudinal study was carried out at the National Institute of Perinatology, Mexico City during 2013–2014. The study protocol was approved by the Institutes' Research and Ethics Committees. A cohort of 92 non‐anemic pregnant women was followed. According to the WHO BMI categories, 39 women were classified as obese (Ob) (prepregnancy BMI ≥ 30) and 53 as with adequate weight (AW) (prepregnancy BMI 18.5–25) using self‐reported pregestational weight. Sociodemographic variables were registered at the first study visit. Weight and fasting blood samples were obtained at weeks 13, 20, 27 and 35 of gestation to determine serum soluble transferrin receptor (sTfR) (ELISA R&D Systems). Weight changes between study visits (weight‐change deltas) were calculated subtracting the measured weight at a given visit from that at the previous visit (Δ1, first to second; Δ2 second to third; Δ3 third to fourth visits). The effect of pregestational obesity on the log‐transformed sTfR was assessed with a General Linear Model (STATA 12), which included gestational age, maternal age and parity as co‐variables. The model was done with and without weight‐change deltas.

Results

In the total sample, age was 31.93±5.52y and 57.6% of women were primiparous; no difference between groups were detected. The weight‐change between visits was lower in Ob than in AW at Δ1 (0.253 ± 0.295 g vs. 0.379 ± 0.189 g, p=0.02), Δ2 (0.285 ± 0.300 g vs. 0.472 ± 0.200 g, p<0.001) and Δ3 (0.312 ± 0.299 g vs. 0.440 ± 0.287 g, p=0.08) respectively.

Concentrations of sTfR increased as pregnancy progressed (coefficient = 0.062, CI95 = 0.034, 0.090, p<0.001), and with higher parity (coefficient = 0.094 95%CI 0.018, 0.171). The increase of sTfR was higher in women with pregestational obesity when weight‐change deltas were included in the model (0.122 95%CI −0.002, 0.248) (p=0.055).

Conclusions

These findings confirm that maternal iron status is affected by parity, and suggests that it may negatively be affected by pregestational obesity when by gestational weight gain is considered.

Support or Funding Information

The study was supported by the Instituto Nacional de Perinatología Isidro Espinosa de los Reyes
Lipid‐Based Nutrient Supplement 2.0: Encapsulation of Oregano Essential Oil to address intestinal parasitic infections in Indian children
Authors: Gaur Shashank , Lopez Emely , Ojha Ankur , Shukla Dharmendra , Patel Pramthesh R , Andrade Juan E
Published: November 2023

Background

Intestinal parasitic infection in one of the main causes of acute undernutrition in children. It alters the intestinal membrane integrity, induces inflammation, undermines nutrient absorption, and causes diarrheal nutrient losses. Oregano essential oil (OEO) is known to reduce intestinal parasitic infections when consumed orally, however, no study exists on their use as an ingredient in lipid‐based nutrient supplement (LNS) formulations. OEO is certified GRAS by FDA for food use; however, its therapeutic use as a supplement has been limited due to its unpleasant flavor, high volatility, and instability to heat and during digestion, all which reduce its effectiveness against intestinal parasites. The aim of this study was to develop a functional LNS containing OEO, which is stable, acceptable and provides controlled intestinal delivery of bioactive.

Methods

Encapsulates of OEO‐beta cyclodextrin (OEO‐BCD) and carvacrol‐BCD (CV‐BCD) (1:1 molar) were prepared using the vacuum desiccator drying (‐20°C) technique and characterized based on encapsulation efficiency, moisture content, morphology, and a 3‐phase in‐vitro digestion stability. CV content was measured using reverse phase HPLC‐UV. LNS containing OEO‐BCD (27.2 mg encapsulate/ 20 g LNS) was formulated using Indian staples and ingredients. Discriminatory sensory tests (triangle) were performed with college students (n=58) and low‐income women (n=25), who were mothers of children <5 y age living in Mehsana, India to evaluate differences between LNS with and without bioactive ingredient (OEO‐BCD only).

Results

Moisture content of the dried encapsulates ranged from 9.6 to 10.2%. Encapsulation efficiency was 73.2±3.5% and 87.2±2.6% for OEO‐BCD and CV‐BCD, respectively. CV content of OEO was 0.58±0.01g/mL. Encapsulate complexes were stable throughout the oral, gastric and intestinal phases where CV release ranged from 7.7–11.65% and 10.71–14.86% for BCD‐CV and BCD‐OEO respectively. Unencapsulated OEO and CV (p<0.05) were labile to digestion and consistently degraded in the gastric phase at 34.9±3.4% and 41.6±2.8%, respectively, compared to the other phases. Sensory test with student and women revealed no differences (p>0.05) in color, aroma, and taste between LNS with and without BCD‐OEO complexes.

Conclusion

Functionalization of LNS with encapsulated OEO is feasible based on in vitro stability and sensory studies. Future studies will address effects of functional LNS on parasite outcomes.

Support or Funding Information

UDSA ‐ Hatch ILLU‐698‐904 | Indian Council of Agricultural Research International Fellowship, India | Office of International Programs, University of Illinois, Urbana‐Champaign
Zinc (Zn) Absorption from Micronutrient Powders (MNP) Is Low in Bangladeshi Toddlers with Environmental Enteric Dysfunction (EED)
Authors: Krebs Nancy F , Long Julie , Mondal Prasenjit , Westcott Jamie , Miller Leland , Islam Munir , Ahmed Mondar , Mahfuz Mustafa , Ahmed Tahmeed
Published: November 2023

Objective

EED has been suspected to impair absorption of micronutrients, including Zn, due to the abnormal small bowel mucosa. MNP interventions with 5 mg/sachet have generally not impacted Zn‐associated outcomes. The objective of this study was to compare Zn absorption across a wide range of MNP Zn doses in young children at risk of EED.

Methods

Bangladeshi children 18–24 mo old, with and without EED (by lactulose:mannitol test) were randomized to MNP with 0, 5, 10, or 15 mg (10 subjects/EED group/dose). Outcomes by EED status included fractional Zn absorption (FAZ) from MNP meal plus from unfortified meals, measured with stable isotopes by urine dual isotope tracer ratio method; total dietary Zn intake (TDZ, mg/d) by duplicate diet collections; and total absorbed Zn (TAZ, mg/d) by FAZ x TDZ. TAZ data were applied to saturation response model (SRM), and to estimated physiologic requirement (EPR, = 0.74 mg/d).

Results

For the first 49 subjects, combined EED groups, mean (SD) TAZ for each MNP dose was: 0.52 (0.29), 0.72 (0.33), 0.96 (0.48), and 1.07 (0.44), respectively (p=0.006). Absorption followed SRM pattern, but overall curve was lower at all MNP doses than the curve for normal 9 month old breastfed U.S. infants (Figure). Only the mean TAZ for 10 and 15 mg MNP doses exceeded the EPR.

Conclusion

Results support impaired Zn absorption in young children risk for EED. Final data analysis will determine whether differences are detected between EED and non‐EED conditions. The results suggest a benefit of a Zn dose in MNP of > 5 mg.

Support or Funding Information

Sponsored by the Bill & Melinda Gates Foundation OPP1113134