bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substrate Synthesis and Inhibitor Design for Synthetase DesD from Streptomyces coelicolor
Authors: Smolarski Brittany A. , Adetyan Hasmik Jasmine , Kingsbury Jason S. , Hoffmann Katherine M 
Published: November 2023

The increasing incidence of drug‐resistant microorganisms poses one of the most severe public health threats to modern society, sparking the need for entirely new classes of antibiotics. Because iron scavenging is vital for pathogen respiration and DNA synthesis, microbial siderophores synthesis represents a large class of previously understudied antiobiotic targets. Desferrioxamine siderophore synthetase DesD catalyzes the ATP‐dependent macrocyclization of ω‐aminocarboxylic acid‐derived cyclodimeric siderophores, and is a model enzyme for NIS synthetases, increasingly associated with bacterial virulence. With aid of x‐ray crystallography and isothermal titration calorimetry, the Hoffmann lab is working to characterize the structure and function of DesD.Collaboration with the Kingsbury group has facilitated organic synthesis of substrates and substrate analogs towards this characterization of DesD. Optimal syntheses of DesD substrates and substrate analogs was successfully accomplished utilizing condition and catalyst screening, gas chromatograph assay, and standard Schlenk technique and glove box handling of compounds. Purity was confirmed using 1H NMR, 13C NMR, IR, and elemental analysis. We are currently employing these substrates in crystallographic structural work and binding studies of DesD to characterize the binding interactions. The utility of this work will be realized in the design and direct synthesis of inhibitors of DesD, which will then provide a basic framework for generation of a new class of antibiotics focused on siderophore inhibition.
Design of a Genetically Encoded 4Fe‐4S Cluster Sensor
Authors: Glynn Calina , Grossman John D , Perlstein Deborah 
Published: November 2023

Iron sulfur cluster biosynthesis is essential in all organisms, but there are currently few cell‐based assays to monitor the cluster assembly and transfer process in vivo. Here we describe our work toward development of a genetically encoded [Fe4S4] cluster sensor that could be used to probe cluster dynamics in vivo. Our sensor design utilizes the cluster driven dimerization of the E. coli transcription factor Fumarate and Nitrate Reductase (FNR) to provide a FRET based readout of cluster binding levels. A fluorescent FNR was developed via genetic fusion of a SNAP‐tag to FNR's C terminus. The SNAP‐tag can be covalently labeled with a variety of fluorophores. We have purified the FNR‐SNAP fusion, verified it can be labeled with fluorophores, and determined that the fusion is monomeric in the absence of [4Fe4S]2+ cluster. With a cluster stabilizing mutant we have demonstrated that the fusion is capable of binding cluster. However, after fluorescent labeling of a mutant known to induce dimerization in the absence of cluster with fluorophores capable of a FRET interaction we were unable to detect a measurable FRET signal. Further analysis of the dimerization mutant implied that the SNAP tag in its current position interferes with the efficiency of dimerization. Current work is focused on minimizing the distance between labels via deletion of FNR's DNA binding domain.Support or Funding InformationBoston University UROP
Lectin‐based methods for in vivo characterization of C‐mannosylated proteins
Authors: Kegley Nicholas Ryan , Naylor Shelley , Rakus John 
Published: November 2023

Glycosylation functions in a variety of ways including ligand recognition and to ensure only proteins which are properly folded are transferred to the Golgi apparatus. Protein‐glycan structures resulting from glycosylation can be differentiated based on the nature of their sugar‐peptide bonds including O‐linked, N‐linked, and the less commonly detected C‐linked. C‐mannosylation is a form of glycolysis in which mannose is attached via a carbon‐carbon bond with the first tryptophan in trp‐x‐x‐trp sequences. Many proteins that are instrumental in the process of inflammation have been found to possess C‐mannosyltryptophan. Lectins are non‐immunological carbohydrate‐binding proteins. Galanthus nivalis lectin (GNL) has been shown to recognize C‐mannosyltryptophan in addition to α1,3‐ and α1,6‐mannose. Our objective is to use GNL pulldowns to isolate and characterize C‐mannosylated proteins from cells grown in culture. Prior PNGaseF treatment removes competing oligomannose structures allowing for C‐mannosyltryptophan‐specific elution of glycoproteins. We have applied this technique to probe C‐mannosylated proteins from B35 glioblastoma cells as well as lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages. GNL‐eluted proteins are lectin blotted with GNL (C‐linked glycosylation positive probe) and ConA (C‐linked glycosylation negative probe). Organization of the neocortex relies heavily on the regulation of neuronal migration. The migration of neurons is dependent on proteins with the C‐mannosylation modification for guidance cues. Proper neurite migration prevents neurological defects in humans. The understanding of the modification in neurite migration is significant because cell migration is an important step in the development of the nervous system. Our objective was to isolate and sequence via mass spectrometry C‐mannosylated proteins that may be involved in this guidance system. An interaction between C‐mannosylated proteins and heat shock protein 70 (Hsp70) has been found to increase the production of TNF‐α in macrophages, which can result in a strong inflammatory response, potentially leading to septicemia if unregulated. The goal of this project is to characterize the interaction between Hsc70 and this heretofore unidentified C‐mannosylated protein. Both LPS and non‐LPS treated cells are grown in parallel and collected at 0, 2, 24, and 48 hour time periods. GNL‐pulldowns were then used to isolate C‐mannosylated proteins and anti‐Hsc70 immunoprecipitation was utilized to isolate all Hsc70‐binding proteins.Support or Funding InformationMarshall University College of Science,West Virginia Higher Education Policy Commission,West Virginia Space Grant Consortium
De Novo Synthesis from Tryptophan in the Absence of a QPRTase Homolog Contributes to NAD+ Biosynthesis in C. elegans
Authors: McReynolds Melanie R. , Wang Wenqing , Holleran Lauren , Hanna‐Rose Wendy 
Published: November 2023

Nicotinamide adenine dinucleotide (NAD+) biosynthesis has proven to be an attractive and promising therapeutic target for influencing health‐span and obesity‐related phenotypes as well as tumor growth. It's a necessity to elucidate exactly how manipulating NAD+ biosynthetic pathways can lead to therapeutic benefits to fully utilize this target for drug discovery. The goal of my research is to understand how NAD+ homeostasis is maintained to support its core metabolic roles and its signaling and regulatory roles involving NAD+ consumers. It's been reported in the literature that C. elegans lack the de novo NAD+ biosynthetic pathway because quinolinic acid phosphoribosyltransferase (QPRTase) is not present in the genome. However, all genes coding for the key enzymes required for production of quinolinic acid (QA) from tryptophan are present in the C. elegans genome. Therefore, we hypothesized that de novo synthesis from tryptophan in C. elegans contributes to NAD+ biosynthesis. In order to investigate if de novo synthesis is indeed active in the absence of the QPRTase homolog, we first asked if QA gets incorporated into NAD+ in C. elegans. Using stable isotope/mass spectrometric flux analysis, I observed that label incorporated into QA was subsequently incorporated into NAD+ as well; supporting the notion de novo NAD+ synthesis is active. I further hypothesized that if QA were an endogenous precursor for NAD biosynthesis, then blocking this pathway would compromise NAD+ levels in vivo. Target metabolomics revealed a decrease in NAD+ and QA levels when this pathway was blocked. This data supports the hypothesis that this pathway is active and required for maintenance of global NAD+ levels. Next, I investigated if supplementation with QA could reverse a phenotype associated with NAD+ deficiency. Consistent with the previous results, supplementation with QA rescued the developmental phenotype and increased NAD+ levels in mutants that lack salvage NAD+ biosynthesis. This evidence suggests that boosting this pathway can lead to homeostatic mechanisms when salvage synthesis is blocked, further supporting its core role in maintaining NAD+ homeostasis. Finally, we've identified key candidate enzymes that may play the role of the missing QPRTase in C. elegans. Using genetics, I am currently investigating the role of these potential enzymes in NAD+ de novo synthesis. This data will outline the mechanisms by which tryptophan de novo synthesis is functioning in C. elegans. This work has implications for efforts to therapeutically target individual NAD+ biosynthetic pathways providing key insight into compensatory homeostatic mechanisms.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health Grant GM086786 (to W. H.‐R.).
Sequestration‐mediated Downregulation of De Novo Purine Biosynthesis by AMPK
Authors: Schmitt Danielle L , An Songon 
Published: November 2023

Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We speculated that AMP‐activated protein kinase (AMPK), which downregulates ATP‐consuming biosynthetic pathways of lipids and carbohydrates, would be a regulator for de novo purine biosynthesis. To understand the functional role of AMPK in the regulation of de novo purine biosynthesis, we have investigated the impact of small molecule‐mediated AMPK activation on de novo purine biosynthesis under fluorescence single‐cell microscopy. While monitoring the subcellular localizations of green fluorescent protein‐tagged pathway enzymes in HeLa cells, we identified that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), was found to associate into cytoplasmic granules, thus self‐assemblies, after treatment with small‐molecule AMPK activators. In addition, subsequent biochemical studies revealed that the cellular levels of purine metabolites were decreased, whereas the ATP levels were conserved upon stimulation of AMPK‐promoted FGAMS self‐assembly. Collectively, we demonstrate that downregulation of de novo purine biosynthesis is controlled by AMPK‐associated signaling pathways through the spatial sequestration of FGAMS into the self‐assembly.Support or Funding InformationUniversity of Maryland, Baltimore County and NIH/NIGMS T32GMO66706 (K. Radtke)
Correlation between Tau Accumulation in Brain and Retina of Individuals with Late Stage Alzheimer's Disease
Authors: Bondarenko Vladimir , Skyba David 
Published: November 2023

This study was aimed to determine if there is a correlation between tau accumulation in the brain and retina of individuals in the late stages of Alzheimer's disease. Characterizing the distribution and accumulation of tau proteins in the retina and brain may be important for early diagnosis of neurodegenerative diseases such as Alzheimer's disease.We examined total tau protein accumulation and its aggregates in brain and retinal tissues from aged individuals with Alzheimer's disease.Tissues from the prefrontal cortex, visual cortex and retinas of ten different cadavers were analyzed. We utilized cryosectioning to prepare tissue sections for immunostaining. Tissues obtained from individuals without a history of Alzheimer's disease or other types of dementia were used as negative controls. Detection of tau was performed using a primary antibody against human tau protein, followed by a universal secondary antibody.Although tau accumulation was greater than controls for both brain tissues, the density was higher in the prefrontal cortex. We also found an increase in tau content in the retinas from individuals with Alzheimer's disease compared to that of individuals without neurodegenerative disorders.These data demonstrate that neurodegenerative changes leading to accumulation of tau aggregates in the brain coincide with increased tau content in the retina. This raises the intriguing possibility that there may be a similar correlation in individuals during the early stages of Alzheimer's disease. Thus, future studies will concentrate on the accumulation of tau in the brain and retina of individuals during the earlier stages of Alzheimer's disease.Support or Funding InformationTouro University Nevada internal research grant.
Hyperglycemia is exacerbated by dysregulation of hepatic SREBP1c‐CRY1 signaling pathway
Authors: KIM JAE BUM , CHOI WON GUN , JANG HAGOON , LEE GHA YOUNG , LEE GUNG , JEON YONG GEUN 
Published: November 2023

SREBP1c is a key transcription factor of lipogenesis to store excess energy in postprandial state. Although SREBP1c appears to be involved in suppression of hepatic gluconeogenesis, the molecular mechanism(s) by which insulin‐activated SREBP1c could repress hepatic gluconeogenesis is not thoroughly understood. Here, we demonstrate that CRY1 activation by insulin‐induced SREBP1c led to decrease hepatic gluconeogenesis through FOXO1 degradation. In accordance with these, SREBP1c−/−and CRY1−/− mice showed higher blood glucose than wild type (WT) mice during pyruvate tolerance test, accompanied with enhanced expression of PEPCK and G6Pase genes. Moreover, CRY1 promoted degradation of nuclear FOXO1 by MDM2, one of the ubiquitin E3 ligases, by enhancing binding of FOXO1 and MDM2. Although hepatic SREBP1c failed to upregulate CRY1 expression in db/db mice, overexpression of CRY1 led to attenuate hyperglycemia through reduction of hepatic FOXO1 protein as well as gluconeogenic gene expression. Collectively, these data suggest that insulin‐activated SREBP1c downregulates hepatic gluconeogenesis through CRY1‐mediated FOXO1 degradation and thereby dysregulation of SREBP1c‐CRY1 signaling cascade in liver could confer hyperglycemia in diabetic animals.Support or Funding InformationThis work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT and Future Planning; 2011‐0018312)
Functional and Behavioral Consequences of Axial Stability in Primates and Other Mammals
Authors: Granatosky Michael Constantine 
Published: November 2023

A stable thoracolumbar region found in some arboreal mammals and certain primates has been proposed as advantageous for maintaining stable back postures during antipronograde locomotion, which is any form of movement in which the forelimbs and/or hindlimbs are loaded “in tension.” However, little data exist testing the inferred link between osteological features cited as enhancing whole‐body stability and the frequency of antipronograde locomotion within an animal's locomotor repertoire. To fill this gap this study uses multivariate methods to compare vertebral morphology of primate and non‐primate mammals with varying levels of antipronograde locomotion within their locomotor repertoires. Overall patterns suggest that species that commonly use antipronograde locomotion exhibit thoracolumbar stability related to osteological features that reduce potential intervertebral spaces. As an additional test of the inferred link between axial rigidity and behavioral stability, this study contrasted the frequency of cantilevering and bridging of Caluromys philander and Loris tardigradus (species that commonly use antipronograde locomotion), to Monodelphis domestica and Cheirogaleus medius (species that rarely use antipronograde locomotion) on a raised horizontal pole and terminal branch experimental set‐up. We observed C. philander cantilevering and bridging significantly more often (55% of observed events) than M. domestica, which never cantilevered or crossed any arboreal gaps (P <0.001). No difference in cantilevering frequency was observed between L. tardigradus and C. medius, but the duration of cantilevering bouts were significantly greater in L. tardigradus (4.24 sec ± 2.11 vs. 2.35 sec ± 1.71; P < 0.01). These data suggest that bridging, cantilevering, and other antipronograde behaviors likely benefit from a stable trunk. However, disparate clustering of antipronograde species based on multivariate statistics suggests that no single adaptive suite characterizes the axial skeleton of these mammals. From this, we can infer that different functional solutions may have evolved to meet the similar mechanical challenges imposed by antipronograde positional behaviors.Support or Funding InformationThis research was support by the National Science Foundation's Graduate Research Fellowship Program and the Force and Motion Foundation.
Proteome‐wide structural analysis of PTM hotspots reveals large body of putative regulatory elements with predicted biological function
Authors: Dewhurst Henry , Torres Matthew 
Published: November 2023

Post‐translational modifications (PTMs) have repeatedly been shown to play vital roles in regulation of protein behavior through modulation of protein‐protein binding and recognition site interactions, allosteric regulation, and conformational switching. SAPH‐ire (Structural Analysis of PTM Hotspots) has been used effectively to reveal novel PTM regulatory elements in G protein families (Dewhurst et al., 2015). Here, we proceed by applying SAPH‐ire to the comprehensive set of eukaryotic protein families containing experimental PTM and 3D structure data. This experiment captures 1,864 protein families and includes more than 60,756 unique PTM sites organized into 40,493 PTM hotspots, of which 2,659 possess known biological function (data obtained from PhosphoSitePlus), analyzed across 51,000 crystalized molecular structures representing 1,080 different species. This PTM data set represents the largest quantitative structural analysis of PTMs to date, yet less than 1/3 of the publically available eukaryotic PTM sites available at the time of investigation. PTM hotspots with a known biological function undergo up to 6‐fold enrichment using SAPH‐ire ranking methods – consistent with pilot studies (Dewhurst et al., 2015). Moreover, a preliminary survey revealed greater than 90% of top‐ranked hotspots lacking functional evidence in our input data could be linked to biological function by in‐depth literature searches. Thus, ~10% of PTM hotspots predicted to be biologically impactful have yet to be verified experimentally and represent putative regulatory elements. Taken together, these results highlight SAPH‐ire's value as a predictive tool for the identification of biologically important PTMs.Support or Funding InformationThis work was funded by the Georgia Tech School of Biology (M.T.) as well as the National Institutes of Health grant 4R00 GM094533‐03 (to M.T.).
Determinants of Gα12‐specific binding common to AKAP‐Lbc and p114RhoGEF
Authors: Garcia Ricardo Daniel , Towne Autumn Lynn , Cavagnini Kyle S. , Meigs Thomas E. 
Published: November 2023

AKAP‐Lbc and p114RhoGEF are guanine nucleotide exchange factors (GEFs) that activate the small G protein Rho. These proteins lack a RGS‐homology (RH) domain that is found in a specific subgroup of RhoGEFs that serve as downstream effectors of heterotrimeric G proteins of the G12/13 family. AKAP‐Lbc plays a crucial role in cardiomyocyte fibrosis and cardiac development, and despite lacking an RH domain, is a binding partner of Gα12. In previous work, we identified a Gα12‐binding region near the C‐terminus of AKAP‐Lbc, and found this region to share close homology with a p114RhoGEF region that we also demonstrated to interact with Gα12. To identify determinants of Gα12 binding within these regions of AKAP‐Lbc and p114RhoGEF, we engineered charge‐substitution mutants at residues common to both RhoGEFs, and located several key amino acids that participate in Gα12 interaction. We also identified a mutant of Gα12 that shows impaired binding to both AKAP‐Lbc and p114RhoGEF, suggesting a common structural motif in the Gα protein for engaging these targets. In addition, we found AKAP‐Lbc and p114RhoGEF to preferentially bind Gα12 in comparison to Gα13, a finding that distinguishes these RhoGEFs from others reported as Gα13‐specific targets. Also, in cell culture assays measuring serum response factor (SRF) mediated transcriptional activation by mutationally activated G12/13, epitope‐tagged G protein binding domains of AKAP‐Lbc and p114RhoGEF selectively blocked Gα12 signaling to SRF. Furthermore, a dominant‐negative, constitutively GDP‐bound variant of Gα12 was more potent than dominant negative Gα13 in hindering serum‐induced p114RhoGEF stimulation of SRF signaling. These results define a Gα12‐selective binding region in AKAP‐Lbc and p114RhoGEF, and implicate p114RhoGEF as an effector of Gα12 mediated cell growth signaling.Support or Funding InformationWe acknowledge support from the GlaxoSmithKline and C.D. Spangler Foundations, UNC Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.
GPR161 and smAKAP Are Membrane‐bound PKA‐RI‐selective AKAP
Authors: Lu Tsan‐Wen , Ilouz Ronit , Stefan Eduard , Taylor Susab 
Published: November 2023

A Kinase Anchoring Proteins (AKAPs) serve as scaffold proteins that target the PKA isoforms to distinct parts of the cell. The hallmark motif that defines an AKAP is an amphipathic helix that docks with high affinity (2–7 nM) onto the N‐terminal helical dimerization/docking domain of the PKA regulatory subunits. While most AKAPs are specific for RII subunits and target PKA, along with other signaling proteins, to channels, transporters and receptors, a few AKAPs are dual specific and can bind to both RI and RII subunits.Some like the small membrane anchored AKAP (smAKAP) bind directly to membranes and are highly specific for RI subunits (Kd = 5 nM for RIs vs. 50 nM for RIIb). We recently discovered AKAP motif that is embedded in the C‐terminal tail of GPR161, an orphan G Protein‐Coupled receptor that is known to be associated with cilia. It is the first discovered G protein that has AKAP motif. This AKAP shows absolute high affinity selectivity for RI (Kd = 6 nM).Structures of the smAKAP bound to the dimerization domain of RIa define the features that convey high affinity binding and specificity to the RI subunits and these features are conserved in the GPR161 AKAP based on peptide arrays. A unique feature of these RI‐specific and/or selective AKAPs is an extended amphipathic helix that caps the two disulfide bonds in the RI subunits. These disulfide bonds are specific feature of the RI subunits that allow them to function as redox sensors.Support or Funding InformationNIH DKS4441 to SST