bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of Herpud1 during osteoclast and osteoblast differentiation
Authors: Quiroga Clara , Da Silva Luan Americo , Mansilla Georthan , Oyarzún Ingrid , Bustamante Mario , Castro Pablo , Verdejo Hugo 
Published: November 2023

Bone is a dynamic tissue characterized by its hardness, rigidity, and high power of regeneration and repair. Its integrity is the result of balance among synthesis mediated by osteoblasts and resorption mediated by osteoclast and the the cross‐talk between them is key to maintain tissue homeostasis.Multiple extra and intracellular signals play an important role in differentiation and function of osteoblasts and osteoclast. Protein quality control (QC) mechanisms is a newly discovered actor in osteoblastogenesis, which requires the activation of proteins involved in endoplasmic reticulum (ER) stress response, particularly the PERK/EIF2AK3‐eIF2A‐ATF4 axis. In contrast, the resorptive phenotype of osteoclast requires activation of macroautophagy machinery.To maintain cellular homeostasis and control the increase of proteotoxic aggregates into the ER, cells activate a program of response (Unfolded Protein Response or UPR) that include the reduction of general protein synthesis, an increase in chaperones synthesis and an increase in the ER‐associated degradation (ERAD).Herpud1, an ER‐membrane integral protein, is highly expressed in response to ER‐stress and is involved in the regulation of ERAD protein complex. Recently we described that a reduction in Herpud1 levels promotes an increase in autophagic flux, showing that this protein negatively controls this mechanism.In function of these antecedents, we aim to assess the role of Herpud1 during the osteoblast and osteoclast differentiation. Pre‐osteoblast MC3T3 and pre‐osteoclast RAW264.7 cell lines were treated with different ER stressors and autophagy inducers in order to compare cellular responses and were maintained in differentiation medium for 3, 7 and 14 days. In case of MC3T3, cell differentiation was evaluated through increases in calcium deposits, alkaline phosphatase (ALP) activity and expression of gene markers of differentiation like RUNX2, OPN, OCN. On the other hand, differentiation of RAW264.7 was assessed by expression and activity of Tartrate‐resistant acid phosphate (TRAP), MMP9 and others. The levels of Herpud1 were determinated through western‐blot and real time PCR.Our results show that Herpud1 levels increase in response to ER‐stressors. Although in initial stages of differentiation (12, 24 h) Herpud1 increases in both cellular lineages, long times of differentiation showed a divergent regulation in its expression. Herpud1 increases during osteoblastogenesis and decreases during osteoclastogenesis, suggesting that Herpud1 has a differential role in osteoblasto‐ and osteoclastogenesis, and could be a candidate to regulate the bone turnover.Support or Funding InformationThis work is supported by FONDECYT 11140470 (CQ), FONDECYT 1150359 (HV) and FONDAP 15130011 (PC, HV).
N‐end‐rule‐mediated Degradation of the Proteolytically Activated Form of PKC‐theta Kinase attenuates its Pro‐Apoptotic Function
Authors: Eldeeb Mohamed Ali , Esmaili Mansoore , Fahlman Richard 
Published: November 2023

Cellular stresses and signalling that lead to the initiation of apoptotic pathways often result in the activation of caspases or calpains which in turn leads to the generation of proteolytically generated protein fragments with new or altered functions. Mounting number of studies reveal that the activity of these proteolytically activated protein fragments can be counteracted via their selective degradation by the N‐End Rule pathway. Here we investigate the proteolytically generated fragment of the PKC theta kinase, where we report the first study on the stability of this pro‐apoptotic protein fragment. We have determined that the pro‐apoptotic cleaved fragment of PKC‐theta is unstable in cells as its N‐terminal lysine targets it for proteasomal degradation via the N‐end rule pathway and this degradation is inhibited by mutating the destabilizing N‐Termini, knockdown of the UBR1 and UBR2 E3 ligases. Tellingly, we demonstrate that the metabolic stabilization of the cleaved fragment of PKC‐theta or inhibition of the N‐end rule augments the apoptosis‐inducing effect of staurosporine in Jurkat cells. Notably, we have demonstrated that the cleaved fragment of PKC theta, per se, can induce apoptotic cell death in Jurkat T‐cell leukemia. Our results expand the functional scope of N‐end rule pathway and support the notion that targeting N‐end rule machinery may have therapeutic implications.Support or Funding InformationMohamed Eldeeb is supported by Alberta‐Innovates Technology Futures (AITF) scholarship.
Bioinformatic and Functional Analysis of Venom from the Jewel Wasp Ampulex compressa
Authors: Arvidson Ryan , Kaiser Maayan , Pan SongQin , Libersat Frederic , Adams Michael E 
Published: November 2023

The parasitoid jewel wasp Ampulex compressa exploits its host, the American cockroach Periplaneta americana, as a food source for its young. The host is subdued through direct envenomation into the central nervous system, inducing a days‐long “zombie‐like state” referred to as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions intact. This condition is reversible, provided that egg deposition is prevented. To facilitate elucidation of biochemical mechanisms underlying venom‐induced hypokinesia, we developed a comprehensive inventory of the venom by combining: 1) next‐generation RNA sequencing, transcriptomic analysis of venom gland components, and 2) proteomics of venom apparatus extracts and whole milked venom via mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology. These analyses yielded a total of 201 proteins and peptides in the venom, 105 with Pfam domains, 146 similar to other proteins in the NCBI‐nr or Uniprot databases, and 45 novel proteins absent in global databases. A. compressa venom proteins include members of the M13 family of proteases (11), phospholipase A2 isoforms (10), along with hyaluronidase, chitinase, icarapin, venom acid phosphatase, serpins, and signaling peptides including tachykinin, corazonin, and myosuppressin. Differential expression analysis and proteomics of the two major tissues of the venom apparatus ‐ venom sac and venom gland ‐identified tissue‐specific expression of key venom components.We show that A. compressa venom tachykinins (AcVTK) are able to activate the cockroach brain tachykinin receptor with high affinity (EC50 ~1 nM) in vitro. Injection of synthetic AcVTK into the subesophageal ganglion, an established target of the wasp venom, increases escape response threshold for one hour post‐treatment. These results indicate that tachykinin may participate in the behavioral changes resulting from envenomation of cockroach cerebral ganglia.We isolated and characterized two novel, paralogous venom sac peptides found in milked venom. These peptides are alpha‐helical, amphipathic, and constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin‐1, into the subesophageal ganglion of cockroaches resulted in a short‐term increase in escape threshold.Our findings show that the venom of A. compressa is a rich collection of both well‐known and novel, pharmacologically diverse compounds. Our study provides new and comprehensive information on A. compressa venom contents that inform hypotheses regarding its mode of action.
Characterization of retinal thickness measurements and their relationship to visual acuity in progressing cases of dry macular degeneration
Authors: Klein Brandon J. , Khadjavi Lily S. 
Published: November 2023

Dry macular degeneration has been linked to thinning of the retinal tissue layers bound between the internal limiting membrane and retinal pigment epithelium (ILM‐RPE). Development of optical coherence tomography (OCT) technologies has introduced a quick, non‐invasive method of measuring the ILM‐RPE thickness in a clinical environment. In this study, we analyzed clinical data including OCT measurements and standard exam data to investigate the relationship between retinal characteristics and visual acuity in 46 dry macular degeneration patients, ranging in age from 47 to 94 years. Strong positive correlations were noted between visual acuity and quantitative, qualitative, and longitudinal OCT measurements. In particular, a multiple linear regression model using only OCT outputs as explanatory variables could predict the patient's visual acuity.Support or Funding InformationLoyola Marymount University Summer Undergraduate Research Program 2015
Syntrophic metabolism of vitamins and amino acids in gut microbial community as revealed by in silico genomic analyses
Authors: Khoroshkin Matvei Sergeevich , Rodionov Dmitry 
Published: November 2023

Metabolite exchange is an important process shaping the structure of microbial communities. However, very little is known about crossfeeding by specific nutrients between microbial community members. Among micronutrients, vitamins of B‐group are of high significance as they are precursors of nearly all metabolic coenzymes that are universally essential in all forms of life. Amino acids are building blocks of proteins and peptides, and also serve as precursors of many essential metabolites including nucleotides, cofactors, etc. Many members of microbial communities are not capable to synthesize some vitamins or amino acids (auxotrophs), while other members possess complete biosynthetic pathways for these nutrients (prototrophs). Therefore, vitamin and amino acid exchange between auxotrophs and prototrophs can be an important factor shaping microbial community structure.We used the metabolic subsystem approach implemented in SEED genomic platform to study potential metabolite exchange between bacteria that inhabit human gastrointestinal tract. We reconstructed metabolic pathways for synthesis of 8 B‐vitamins and 20 amino acids in 430 human gut bacteria with genomes sequenced by Human Microbiome Project. We also analyzed genomic distributions of known uptake transporters for vitamins and amino acids. The inferred vitamin and amino acid proto‐/auxotrophic phenotypes and predicted transporters allowed us to classify the studied organisms with respect to their biosynthetic and transport capabilities. The studied bacteria showed high level of conservation of proto‐, auxotrophic phenotypes on the taxonomic level of species. Incomplete biosynthesis pathways for some vitamins, such as biotin, thiamine and pantothenate, suggest certain vitamin deficiencies can be alternatively supplemented by their metabolic precursors (e.g. dethiobiotin, thiazole, pantoate). Overall, vitamin auxotrophic phenotypes are much more abundant in the human gut microbiota, whereas the larger number of studied bacteria is capable of de novo synthesis of all 20 amino acids. Histidine and tryptophan have the most variable phenotypes within the Clostridiales lineage suggesting that crossfeeding by these amino acids is important for gut community. In summary, the obtained in silico reconstructions of vitamin and amino acid metabolic pathways contribute to our understanding of metabolic crossfeeding processes in human gut microbiome.Support or Funding InformationThis research was supported by the Russian Science Foundation (grant #14‐14‐00289) and National Institute of Health (GM108527).
Purification of Truncated Wild‐Type and Mutant Recombinant Lipoprotein Lipase and Investigating Their Ability to Regulate ABCA1
Authors: Madhwani Kimberly Rose , Medh Jheem D. 
Published: November 2023

BackgroundLipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides in extracellular lipoprotein particles. It is expressed by macrophages and secreted to the cell surface. The LPL molecule is comprised of a large amino‐terminal domain that contains its lipolytic activity, and a small carboxyl‐terminal domain that functions as an adapter between lipoprotein receptors/cell surface proteoglycans and apolipoproteins. From previous studies, we discovered that silencing the LPL gene in THP‐1 macrophages promotes the expression and function of an atheroprotective cholesterol transporter, ABCA1. Thus, the objective of this project is to synthesize recombinant LPLC, a truncated molecule lacking the catalytic domain; and LPLC‐WW, a mutant carboxyl‐terminal domain that is unable to bind lipoproteins. Our long‐term goal is to determine the molecular components of LPL that contribute to its ability to modulate ABCA1 expression.MethodsRecombinant glutathione‐S‐transferase (GST) fusion proteins were prepared using pGEX‐2T plasmids. These plasmids, containing the genes for the WT and WW LPLC domain were obtained as a gift. pGEX‐2T LPLC‐WT and LPLC‐WW plasmids were transformed into competent E.coli cells and plated on LB+ampicillin agar plates. Viable colonies were picked and inoculated into 2‐ml cultures of 2X‐YTA medium, before being transferred to a large‐batch culture. The larger cultures were induced with 100 mM IPTG for 2–6 hours, pelleted and resuspended with 1X PBS. Resuspended pellets were sonicated at 37º for 10 minutes and solubilized in 20% Triton X‐100. Glutathione Sepharose 4B was used to immobilize the GST fusion proteins while the contaminants were washed away with 1X PBS. Thereafter, the fusion proteins were eluted from a small aliquot of beads by Elution Buffer containing 10 mM Glutathione. The elution of GST‐LPLC was confirmed by 15% SDS‐PAGE. Next, a thrombin mixture was added to the Glutathione Sepharose 4B‐bound fusion proteins, and incubated for 12–16 hours to cleave and separate the LPLC proteins from the immobilized GST. The presence of purified LPLC proteins was confirmed by 15% SDS‐PAGE and Coomassie Blue staining.ResultsBoth LPLC‐WT and LPLC‐WW were obtained free of any contaminants including GST. We obtained 106 μg of LPLC‐WT and 116.7 μg of LPLC‐WW and are continuing to scale‐up the purification.ConclusionsThe pGEX‐2T plasmids are suitable tools for the preparation of GST fusion proteins. The use of thrombin and a glutathione‐sepharose column efficiently removed the GST component to obtain pure protein. The recombinant LPLC‐WT and LPLC‐WW will be valuable reagents for studies on the molecular components required for the modulation of ABCA1 expression.Support or Funding InformationThis work was supported by National Institutes of Health Award SC3GM095413.
Regulating Skeletal Progenitor Cells at the Interface Between Tendon and Bone
Authors: Merrill Amy Elizabeth , Roberts Ryan 
Published: November 2023

The surfaces of bones are adorned with projections that mark their integration into the musculoskeletal system. These structures, known as bone eminences, take on diverse shapes and sizes to form anchoring points for tendons and ligaments. Bone eminences represent the mineralized side of the tendon‐bone attachment unit, which is composed of a transitional connective tissue, graded from tendinous to osseous. As such, the eminence plays a critical role transmitting forces generated by muscle contraction and dissipating stress concentrations across the hard‐soft tissue interface. During development, the tendon‐bone attachment unit descends from a unique pool of Scx+/Sox9+ progenitors that form secondary to those of the primary cartilage anlage and later become segregated into Scx+ tenocytes or Sox9+ chondrocytes of the eminence (Bltiz et al., 2009, 2013; Sugimoto et al., 2013). While many open questions remain about the molecular regulators that demarcate this boundary across the Scx+/Sox9+ progenitor field, chick embryo studies suggest a role for Fibroblast Growth Factor (FGF) signaling (Brent et al., 2003, 2004, 2005). We have uncovered evidence that Fgfr2 regulates cell fate choice during development of tendon‐bone attachment units in the craniofacial complex of mice. We have found that loss of Fgfr2 increases the eminence superstructure by promoting chondrogenic over tenocytic cell fate in the attachment unit progenitors. These findings contribute to the understanding of tendon‐bone attachment unit development and reveal a possible mechanism to explain the constellation of clinical abnormalities of the tendon‐bone interface that are found in the skeletal birth defects caused by gain and loss of FGFR2 function.Support or Funding InformationNIH, NIDCR #1R01DE025222 to A.E.M.March of Dimes, Gene Discovery and Translational Research Grant #6‐FY15‐233 to A.E.M.NIH, NIDCR #T90DE021982 to R.R.
Fluorescent Assay of Ubiquitin Conjugation
Authors: Jackson Cassidy , Berndsen Christopher 
Published: November 2023

Ubiquitination of proteins is a post translational protein modification linked to protein degradation, cell cycle regulation, and DNA damage repair. Many of the functions of ubiquitin and ubiquitin like proteins are still unknown and the catalytic mechanisms of the conjugating enzymes are partially known. The assays of ubiquitin/ubiquitin‐like protein conjugating include observing changes in migration on gel electrophoresis or radioactive tags which are expensive and require special handling. We have optimized a fluorescence based assay that quantifies the cleavage of ATP to AMP by the E1 ubiquitin/ubiquitin‐like protein activating enzyme. This assay permits study of the kinetics of ubiquitin conjugation in a continuous or discontinuous manner and is highly sensitive. We have optimized the conditions for monitoring E2 dependent ubiquitin chain formation in order to determine the mechanism of E1 and E2 enzymes. This assay will help in the understanding of the mechanism of ubiquitin and ubiquitin like protein conjugation.Support or Funding InformationUS‐Israel Bi‐national Science Foundation 2013261
Structural and Functional Substrate Binding in an Iterative Non‐ribosomal Peptide Synthetase Independent Siderophore (NIS) Enzyme from Streptomyces coelicolor
Authors: Hoffmann Katherine M 
Published: November 2023

Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation outside of a ribosome. One type of siderophore synthesis uses Non‐ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases) to form amide bonds (peptide bonds) between metabolic intermediates in NIS siderophore biosynthesis pathways. Recently, primary structure analysis, correlated with known activity, has identified four subtypes (A, A′, B and C), only two of which have been structurally described. While subtype is based on the primary substrate specificity, an additional remarkable kinetic behavior exists in a sub‐category of enzymes whereby multiple iterative bonds may be catalyzed on the same substrate. Prior work in the field has identified a novel protein fold (including a novel ATP binding motif,) but several sub‐types and iterative proteins are yet to be structurally or biochemically described. The iterative activity is correlated with broad substrate specificity, but the binding affinity has never been quantified. In fact, iterative enzyme kinetic parameters have been elusive, as the first published biochemical characterization found the reporters limited the speed of the assays.We are structurally and functionally characterizing a representative iterative NIS enzyme, DesD, from Streptomyces coelicolor, using the methodologies of X‐ray crystallography and isothermal titration calorimetry (ITC). Our results suggest a firm order of binding and significantly more substrate specificity than previously thought. Additionally, the innovative use of calorimetry to measure kinetic parameters has allowed a reporter‐free assay that may be broadly applicable to other processive enzymes. Finally, we are structurally characterizing the apo, partial complex, and full complex of DesD bound to it's high‐specificity substrates, to fully map binding interactions. We hope future work will include the design and synthesis of inhibitors, as well as site‐directed mutagenesis, to probe mechanism of the novel chemistry.
The Structural Basis of Cyclooxygenase‐2 Inhibition by Fenamates
Authors: Xu Shu , Banerjee Surajit , Kudalkar Shalley N , Perry Evan T , Ghebreselasie Kebreab , Marnett Lawrence J 
Published: November 2023

Two cyclooxygenase (COX) isoforms, COX‐1 and COX‐2, catalyze the first two steps of prostaglandin biosynthesis from arachidonic acid (AA) and are the pharmacological targets of non‐steroidal anti‐inflammatory drugs (NSAIDs). Mefenamic acid is one of several NSAIDs that have shown selective inhibition of 2‐arachidonylglycerol (2‐AG) oxygenation by COX‐2 (Prusakiewicz J.J. et al, Biochemistry 2009). While the X‐ray crystal structures of most classes of NSAIDs bound to COX have been solved, the binding mode of fenamates is still unknown. Here we report the structural determination of murine COX‐2 structures complexed with several fenamates and biochemical investigation of the role of individual residues in their inhibition. The crystal structures for the first time revealed that the fenamates are bound in the cyclooxygenase channel in an inverted binding mode similar to those of propionic acid derivatives such as diclofenac and lumiracoxib. The side chains of Tyr‐385 and Ser‐530 form critical H‐bonds with the carboxylic group of the fenamates. The aniline ring substituents of mefenamic acid, tolfenamic acid and flufenamic acid exhibit a preference for the space beneath Leu‐531 as opposed to the side pocket off the active site channel. In contrast, when there is an additional chloride substituent, as in the case of meclofenamic acid, this extra substituent orients toward the side pocket. These observations provide a structural basis for the fact that meclofenamic acid is a slow, tight‐binding inhibitor of COX‐2 with a low IC50 (31 nM) whereas mefenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid are weak, rapidly reversible inhibitors with IC50's greater than 10 μM for AA oxygenation. Strikingly, when 2‐AG is used as the substrate, these weak, rapidly reversible inhibitors become potent inhibitors with IC50's ranging from 20 to 50 nM. In contrast, meclofenamic acid exhibits the same potency for both substrates. Our results provide the first atomic level detail of the interaction between fenamates and COX‐2, structural insights of slow, irreversible and rapid reversible fenamates, and a biochemical and structural foundation for future design and development of substrate‐selective inhibitors of COX‐2.Support or Funding InformationThe work was supported by NIH CA089450 (to L. J. M.).