bluuebunny/crossref_metadata_2025_split huggingface dataset:
Charecterization of a Glycerophosphocholine Acyltransferase (Gpc1), a Novel Enzyme in Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae
Authors: Anaokar Sanket , Lager Ida , Glab Bartosz , Banas Antoni , Stymne Sten , Patton‐Vogt Jana 
Published: November 2023

Phosphatidylcholine (PC) is the major phospholipid found in most eukaryotic membranes. The maintenance of membrane composition through regulated synthesis and turnover of phosphotidylcholine is crucial to cell functioning. We have identified a novel enzyme glycerophosphosphocholine acyltransferase (Gpc1) that expands our understanding of PC biosynthesis and remodeling. Gpc1 acylates glycerophosphocholine (GPC), a product of complete deacylation of PC to Lysophosphotidylcholine (LysoPC), which can subsequently be converted to PC by lysophospholipd acyltransferase, Ale1. The yeast knock‐out collection was screened in order to find the Gpc1‐encoding gene. In vitro assays confirm that Gpc1 is an acyltransferase involved in PC biosynthesis. In addition, metabolic labeling studies performed in various strain backgrounds in which the established pathways for PC biosynthesis were attenuated indicate an in vivo role for Gpc1 in PC biosynthesis. Factors and growth conditions that regulate GPC1 expression are being explained. Gpc1 belongs to a new protein family as it shows no similarities to the known acyltransferases or transacylases. Gpc1 homologs are found in multiple organism including plants and medically important pathogenic fungi, but they are absent in vertebrates. Overall, we have successfully identified Gpc1, a glycerophosphocholine acyltransferase encoding gene that contributes to PC biosynthesis in the cell.Support or Funding InformationNational institute of Health Grant NIH R15 GM104876
The Nem1‐Spo7/Pah1 Phosphatase Cascade is Negatively Regulated by Protein Kinase A
Authors: Su Wen‐Min , Han Gil‐Soo , Carman George M 
Published: November 2023

Yeast Pah1 phosphatidate phosphatase, which dephosphorylates phosphatidate to generate diacylglycerol, catalyzes the penultimate step in the synthesis of triacylglycerol. The translocation of Pah1 from cytosol to ER membrane is essential for its function and is strictly dependent on the nuclear/ER membrane‐associated Nem1‐Spo7 protein phosphatase complex. Dephosphorylation of Pah1 by Nem1‐Spo7 enables its membrane association, and also stimulates its phosphatidate phosphatase activity. Previous work has shown that protein kinase A phosphorylates Pah1 to inhibit its function in lipid metabolism. Here, we showed that Nem1, the catalytic subunit of the protein phosphatase complex, is also phosphorylated by protein kinase A. The kinase phosphorylated Nem1 on multiple serine residues and caused a 35% decrease in its protein phosphatase activity for phospho‐Pah1. The Nem1 synthetic peptides KRNRGSNASEN (Vmax/Km = 2.2 μmol min−1 mg−1 mM−1) and RPRSYSKSELS (Vmax/Km = 94.9 μmol min−1 mg−1 mM−1) that contain Ser‐140 and Ser‐210, respectively, were phosphorylated by protein kinase A. Site‐direct mutagenesis of Ser‐140 and Ser‐210 in Nem1 followed by phosphopeptide analysis showed that Ser‐210 was the major protein kinase A target site. The alanine mutation of Ser‐210 caused a 50% reduction in the in vivo phosphorylation state of Nem1. These data indicated that protein kinase A plays a negative regulatory role on the activity of the Nem1‐Spo7/Pah1 phosphatase cascade.Support or Funding InformationSupported by NIH grant GM050679
Crystal Structure of LCAT Bound to a Small Molecule Allosteric Activator Reveals its Active Conformation
Authors: Manthei Kelly A. , Yang Shyh‐Ming , Chang Louise , Freeman Lita A. , Baljinnyam Bolormaa , Shen Min , Maloney David J. , Remaley Alan T. , Jadhav Ajit , Tesmer John J.G. 
Published: November 2023

There is an inverse relationship between risk for coronary heart disease (CHD) and the level of high density lipoprotein cholesterol (HDL‐C). As CHD is one of the leading causes of death worldwide, there is great interest in mechanisms to raise HDL‐C for treatment of CHD. Lecithin:cholesterol acyltransferase (LCAT) catalyzes the transfer of an acyl chain from phosphatidylcholine to cholesterol to create a cholesteryl ester (CE) in the process of reverse cholesterol transport. The formation of the more hydrophobic CE leads to the maturation of HDL from discoidal to spherical and promotes the clearance of cholesterol from arterial plaques. Mutations in LCAT are known to cause two genetic diseases: fish eye disease (FED) and familial LCAT deficiency (FLD). Individuals with either FED or FLD have low HDL‐C and corneal opacities, while FLD patients ultimately develop renal failure. Recombinant LCAT and LCAT‐activating compounds are being investigated as treatments for both CHD and FLD. However, only recently has any structural information on LCAT been available to guide development of these therapies. Based on recent crystal structures and hydrogen deuterium exchange data, it is now apparent that LCAT has a dynamic lid that is able to protect the LCAT active site from solvent. This lid likely plays a critical role in activation of LCAT by apolipoprotein A‐I, the major protein component of HDLs. We report on a new structure of LCAT bound to an LCAT‐activating compound as well as an acyl intermediate state mimic, isopropyl dodecylfluorophosphonate. The combination of these two ligands promotes a lid‐open conformation. We confirmed that the compound binds to an allosteric site via site directed mutants that both eliminate binding and increase the thermal stability of LCAT. These studies pave the way for the design of rational drugs or improved biotherapeutics that can be used to treat CHD and FLD and augment reverse cholesterol transport. The new structure also allows for a better understanding of the acyl binding site of LCAT and its catalytic mechanism.Support or Funding InformationThis work was supported by US National Institutes of Health (NIH) grants HL122416 and Postdoctoral Fellowship F32HL131288 (K.A.M.).
Energy substrate levels and metabolic changes in skeletal muscle underlie increased activity and improved exercise performance in liver fatty acid‐binding protein null mice
Authors: Xu Heli , Gajda Angela , Zhou Yin Xiu , Fatima Anam , Storch Judith 
Published: November 2023

Liver fatty acid‐binding protein (LFABP, FABP1) binds long chain fatty acids with high affinity, and is abundantly expressed in the liver and small intestine. Although LFABP is thought to function in intracellular lipid trafficking, studies of LFABP null (LFABP−/−) mice have also demonstrated a role in regulation of systemic energy homeostasis. We and others have reported that LFABP−/− mice become more obese than wild‐type (WT) mice upon high fat feeding. Despite increased body weight and fat mass, however, and in marked contrast to the drastic decrease in exercise capacity in high fat‐fed WT mice, LFABP−/− mice were able to maintain their endurance exercise performance, showing an approximate doubling of running distance compared with the WT mice. Therefore, to understand this surprising exercise phenotype, in the present studies we focused on the metabolic alterations in the skeletal muscle secondary to LFABP ablation. The results indicate significantly increased triglyceride (TG) content and glycogen content in the skeletal muscle of LFABP−/− mice. In addition, higher mitochondrial enzyme activities, in both the tricarboxylic acid (TCA) cycle and the electron transport chain, suggest that LFABP−/− muscle has greater energy substrate availability and greater capacity for substrate oxidation compared with WT muscle. Moreover, consistent with the findings that their muscle Pgc1α and Pparα mRNA levels were upregulated, LFABP−/−mice had an increased fatty acid oxidation rate in the skeletal muscle compared with WT mice. Interestingly, however, substrate utilization during exercise showed no differences between LFABP−/− and WT mice, as examined by respiratory exchange ratio, exercise‐dependent decreases in muscle TG and glycogen stores, and 5′AMP‐activated protein kinase activation levels. Thus, the LFABP−/− and WT mice appear to have similar energy production during exercise, indicating that the markedly improved exercise capacity in the LFABP−/− mice is related to their higher muscle substrate levels prior to exercise. Overall, the results show that although it is not expressed in muscle, LFABP ablation in the liver and intestine is associated with alterations in skeletal muscle energy metabolism and energy substrate levels. These changes may be due, in part, to liver‐and/or intestine‐derived mediators that contribute to the greater TG and glycogen availability and increased mitochondrial function, thereby preventing the high fat feeding‐induced decrease in exercise capacity.Support or Funding InformationThis work was supported by National Institutes of Health Grant DK‐38389 from NIDDK, and by funds from the New Jersey Agricultural Experiment Station (to J. S.).
Myopalladin's Role in Cardiac Muscle Function and Disease
Authors: Kadarla Vinay Kumar , Bigge Brae Marie , Beck Moriah Rene 
Published: November 2023

Decades of research have provided fundamental insight into the human heart's structure and function. Yet, most cardiac malformations remain a mystery as scientists and clinicians continue to examine how inherited mutations and aging affect the normal biological functions of proteins associated with cardiac dysfunction. Recently, mutations in the muscle protein myopalladin have been linked to the pathogenesis of cardiomyopathy. Myopalladin is a sarcomeric protein that is thought to have an important role in maintaining sarcomere structure, signaling and regulation of gene expression in response to muscle stress. Myopalladin and palladin belong to a family of closely related immunoglobulin (Ig)‐domain‐containing proteins that have essential, but unclear roles in organizing the actin cytoskeleton. Recent work in the Beck lab has shown that the C‐terminal Ig domains of palladin bind directly to actin and increases both the rate of actin polymerization and the stability of actin filaments. The fact that a number of mutations in myopalladin are located within the analogous actin‐binding region suggests that a disruption in actin regulation may occur in cardiomyopathy. Thus, we hypothesized that myopalldin also binds directly to actin and increases both the rate of actin polymerization and the stability of actin filaments. To study the capability of myopalladin to bind and crosslink actin filaments, co‐sedimentation assays were performed between purified F‐actin and myopalladin Ig domains at various concentrations. The results suggest that Ig3 domain of myopalladin is the minimal domain required for both the binding and bundling of F‐actin. Pyrene fluorescence was used to monitor the polymerization rate of G‐actin in the presence of increasing concentrations of the various Ig domain of myopalladin. Our data reveals that myopalladin increases both the rate and extent of actin polymerization. From our data, we propose that myopalladin is involved in sarcomere structure and function through regulation of actin dynamics. We suggest that myopalladin may act both as a scaffold, binding directly to actin and other actin binding proteins at the Z‐disc, and by regulating actin thin filament turnover.Support or Funding InformationThis project was supported by the Kansas INBRE under grant number P20 GM103418; The WSU Carl and Rozina Cassat Regional Institute on Aging Grant (2016); and Wichita State University.
Designed Proteins Induce Formation of Nanocage‐containing Vesicles
Authors: Votteler Jörg , Ogohara Cassandra , Yi Sue , Hsia Yang , Nattermann Una , Belnap David M , King Neil P , Sundquist Wesley I 
Published: November 2023

Complex biological processes are often performed by self‐organizing nanostructures comprising multiple classes of macromolecules. For example, enveloped virus are built by self‐assembling proteins that package other proteins and nucleic acids, and acquire a lipid envelope by budding through limiting membranes. Viral structural proteins contain the membrane binding, assembly and budding activities necessary to create these extracellular virus particles, which can transduce new cells and deliver their cargoes. These activities underlie the efficacy of viral vectors, which have been explored extensively as therapeutic delivery vehicles. However, the use of viral vectors for therapy can be restricted by their limited packaging capacity, safety concerns, and immunogenicity.To expand the toolbox of candidate delivery systems and to test our understanding of the fundamental requirements for viral biogenesis, we have designed protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles viral assembly and release pathways. We refer to these vesicles as enveloped protein nanocages (EPNs). Robust EPN assembly and release required three elements: membrane binding, self‐assembly, and recruitment of the cellular ESCRT pathway (endosomal sorting complexes required for transport). We have identified 16 different combinations of membrane binding, self‐assembly, and ESCRT recruiting elements that can produce EPNs efficiently, indicating that the strategy is very general. Biochemical and cryo‐electron microscopic analysis of one EPN design, termed EPN‐01, revealed that the protein nanocages precisely matched the structure of the designed 60‐subunit self‐assembling scaffold. EPN‐01 was released from cells within small membrane enclosed vesicles that each contained multiple nanocages. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV‐G) were able to fuse with new target cells, thereby transferring their cargos from one cell to another, as assayed by successful packaging and delivery of a β‐lactamase‐Vpr fusion protein. We are now using these systems to analyze how different variables affect nanocage envelopment and release, and as a starting point for creating new synthetic delivery systems that are based on the viral assembly principles.Support or Funding InformationNIH P50 082545
Regulation of Angiotensin Receptor Trafficking by an Upstream Short Open Reading Frame in the mRNA 5′ Leader Sequence
Authors: Kadam Parnika , Mueller Susette , Ji Hong , Sandberg Kathryn 
Published: November 2023

BackgroundA seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5′ leader sequence (5′LS) of the angiotensin type 1a receptor (AT1aR) mRNA is highly conserved in rats, mice and humans.AimOur goal is to investigate the mechanism by which PEP7 modulates AT1aR signaling cascade.MethodThe start codon of the sORF was mutated at adenine ‐108 (A−108 to T−108) to create E1,2(−108T),3‐AT1aR and cloned into the pEGFP‐N2 plasmid (construct). Human embryonic kidney (HEK) 293 cells were transfected with intact or the disrupted sORF construct. Live cell imaging in the presence of a LysoTracker dye (staining lysosomes), immunostaining and western blot analysis were performed after treatment with Ang II (100 nM).ResultsWhile the rate of vesicle formation after Ang II treatment was decreased by disrupting the PEP7 sORF [t1/2(s): E1,2,3‐AT1aR, 203s vs E1,2(−108T),3‐AT1aR, 328s; p<0.05; n=15], disruption of the PEP7 sORF did not alter the transport of AT1aR‐GFP to lysosomes within the first 25 minutes of Ang II stimulation. Immunostaining suggested that disruption of PEP7 sORF alters Ang II‐induced colocalization of AT1aR‐GFP with β‐arrestin and pERK1/2 with β‐arrestin. Western blot analysis showed that disruption of PEP7 sORF decreased the level of ERK activation in transfected HEK 293 cells. [pERK1/2 /total ERK1/2: E1,2,3‐AT1aR, 1.2 ± 0.1 vs E1,2(−108T),3‐AT1aR, 0.79 ± 0.0002, p<0.05; n=2]ConclusionPEP7 sORF regulates Ang‐II induced AT1aR internalization and signal transduction through the β‐arrestin‐pERK1/2 pathway by increasing ERK1/2 activation.Support or Funding InformationNIH R01‐HL121456 to KS.
Toward Facile Automated Glycan Synthesis: Current Progress and Remaining Challenges
Authors: Pohl Nicola Lucia 
Published: November 2023

Structure/function studies related to the biochemistry and molecular biology of carbohydrates has lagged those of proteins and nucleic acids in part due to the challenges in obtaining structurally well‐defined glycans. Fortunately, a 2012 report by the National Academy of Sciences1 outlining the specific problems has spurred investment by the U.S. National Institutes of Health through its Common Fund2 in developing the chemical methods and overcoming the building block limitations to make automated oligosaccharide synthesis widely available. Interestingly, individual investigators funded through this synthesis program are expected to coordinate and synergize efforts rather than work in isolation and to develop standardized, vetted protocols. This talk will discuss progress to date3 and the challenges still ahead in making a wide variety of discrete oligosaccharide structures readily available through automated methods.Support or Funding InformationNational Institutes of Health (5U01GM116248‐02)
The expanding glycouniverse: diverse glycan modifications in lower eukaryotes
Authors: Wilson Iain B. H. , Yan Shi , Hykollari Alba , Eckmair Barbara , Vanbeselaere Jorick , Paschinger Katharina 
Published: November 2023

The classical view has been that N‐linked oligosaccharides in ‘lower’ eukaryotes (protists, fungi, plants and invertebrates) are simple. However, recent data shows that, other than plants, the diversity of N‐glycan modifications in non‐vertebrate species is extremely high. From our own studies on Dictyostelium, Trichomonas, Penicillium, Acanthamoeba, mosquitoes and other insects, Caenorhabditis and other nematodes, molluscs and planaria, a wide range of new glycan modifications have been demonstrated using an off‐line HPLC/MALDI‐TOF‐MS workflow. Particularly underrepresented in older studies are the ‘charged’ modifications of N‐glycans from lower eukaryotes, such as glucuronic acid, sulphate, phosphoethanolamine, aminoethylphosphonate and phosphorylcholine. In addition, unusual galactosylation, fucosylation and mannosylation events (on the core and bisecting positions) increase the range of possibilities. Thus the concept of ‘simple’ organisms possessing only ‘simple’ glycans is no longer a true reflection of the actual glycosylation capacity of invertebrates. The coming years are certain to result in further glycomic surprises, certainly reflecting the presence of an unexplored treasure chest of Golgi enzymes in the analysed species.Support or Funding InformationThis work has been funded by the Austrian Science Fund (FWF; grants P26662, P25058 and P23922 to A.H., K.P and I.B.H.W.) and by the European Union (GlycoPar Initial Training Network; PITN‐GA‐2013‐608295).
The Human Lectin Galectin‐3 Recognizes Chondroitin Sulfate Proteoglycans (CSPGs) and Sulfated Glycosaminoglycans
Authors: Dam Tarun , Talaga Melanie , Fan Ni , Fueri Ashli , Brown Robert , Bandyopadhyay Purnima 
Published: November 2023

Glycan‐binding proteins are classified in two major but distinct groups: lectins and glycosaminoglycan (GAG)‐binding proteins (GAGBPs). The multifunctional human lectin galectin‐3 (Gal‐3) is a member of the first group. GAGBPs, on the other hand, include a variety of non‐lectin proteins such as growth factors, cytokines, morphogens and ECM (extracellular matrix) proteins. The general structures of the binding sites of lectins, their preferred glycan structures and their mode of action are fundamentally different from those of GAGBPs. A member of one group rarely possesses the characteristics of both groups. Our data, however, show that Gal‐3 is a notable exception to that rule. In the present study, we report several significant findings. First, calorimetric and spectroscopic data show for the first time that Gal‐3 behaves like a GAGBP. The lectin interacts with unmodified sulfated GAGs (heparin and chondroitin sulfates) and chondroitin sulfate proteoglycans (CSPGs) at physiological pH and NaCl concentration. Second, the binding of sulfated GAGs and CSPGs to Gal‐3 is not a result of non‐specific interactions, rather sulfated GAGs and CSPGs specifically bind to Gal‐3, apparently, via the LacNAc/lactose binding site of Gal‐3. Finally, while heparin and chondroitin sulfate B (dermatan sulfate) are found to be monovalent ligands of Gal‐3, chondroitin sulfate A (CSA), chondroitin sulfate C (CSC) and bovine CSPG engage in multivalent binding with Gal‐3. Such multivalent binding leads to the formation of noncovalent cross‐linked complexes, which is reversible by lactose and different sulfated GAGs. Hill plot analysis of calorimetric data show that the binding of CSA, CSC and bovine CSPG to Gal‐3 is associated with progressive negative cooperativity effects. The present study provides a foundation for discovering new functions of GAGs and CSPGs mediated by Gal‐3 [1].Support or Funding InformationNational Science FoundationMichigan Technological University
Using Recombined RNA Aptamers to Specifically Change O‐GlcNAcylation on a Protein
Authors: Zhu Yi 
Published: November 2023

O‐linked β‐D‐N‐acetylglucosamine (O‐GlcNAc) is an abundant post‐translational modification (PTM) that has been reported on more than 4,000 intracellular proteins. In contrast, there are only two enzymes known to carry out its addition and removal: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). And it remains poorly understood how these enzymes recognize each of their substrates. As a result, researchers are limited in experimental approaches that specifically change O‐GlcNAc levels on a protein, and this is greatly hindering us from dissecting the regulatory roles of O‐GlcNAc. Once called “nucleic acid antibodies”, aptamers are usually short single‐stranded DNA or RNA, which bind their targets with high affinities and specificities. In this research, we developed a method that is capable of changing O‐GlcNAcylation on a specific protein, using recombined RNA aptamers. We performed Systematic Evolution of Ligands by Exponential Enrichment (SELEX), an iterative in vitro selection with a random RNA library, and identified an RNA sequence that recognizes the nucleocytoplasmic isoform of OGT (ncOGT) with high affinity (Kd~50nM). This aptamer binds to the tetratricopeptide repeat (TPR) domain of ncOGT and does not impair its enzymatic activity. We recombined this aptamer with another aptamer that recognizes GFP, and overexpressed the recombined aptamer together with a GFP‐tagged protein in HEK293T cells. Data from Immunoprecipitation (IP) and Western Blot showed that O‐GlcNAcylation on the GFP‐tagged protein was significantly increased in the presence of this recombined aptamer. The interaction between OGT and the GFP‐tagged protein was promoted as well, based on Co‐Immunoprecipitation (Co‐IP) results. Using this method, we were able to specifically increase O‐GlcNAcylation on several GFP‐tagged proteins, including transcription factors and kinases. Taken together, these results suggest recombined RNA aptamers as promising tools to specifically change O‐GlcNAcylation on individual proteins, which will further provide researchers a method to study the regulatory function of O‐GlcNAc with more details.Support or Funding InformationSupported by NIH P01HL107153, R01DK61671 and R01GM116891. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.