bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identifying serine proteases involved in HMPV F cleavage
Authors: Kinder Jonathan Tyler 
Published: November 2023

Human metapneumovirus (HMPV) is a recently identified pneumovirus which causes severe acute respiratory tract infections in children, elderly and immunocompromised patients worldwide. Though this viral pathogen is ubiquitous and responsible for a large number of respiratory tract infection‐associated hospitalizations, there are no vaccines or antiviral therapies other than supportive therapy. HMPV encodes for three surface glycoproteins including the small hydrophobic protein, the attachment protein and the fusion protein (F). The main mediator of viral infectivity is the F protein, which attaches to host cell surface receptors and promotes fusion between the viral and cellular membranes to mediate successful entry. However, in order to mediate viral infection, the HMPV F protein must be proteolytically processed by host serine proteases into a metastable form. TMPRSS2 is a serine protease that has been shown to cleave HMPV F and allow for proper protein function and subsequent infection1. Other serine proteases that are hypothesized to be involved in HMPV F cleavage include HAT, Matritpase, KLK5 and KLK12, all of which have been shown to process influenza hemagglutinin. Using transiently expressed HMPV F in Vero cells, we have verified the requirement of a serine protease to cleave HMPV F. Data on analysis of the ability of both transmembrane and secreted serine proteases to process HMPV F into its fusogenically active form will be presented.Support or Funding InformationUniversity of Kentucky department of cellular and molecular biochemistry
Affinity and Structural Characterization of Human AT Hook Motif Variants
Authors: Dobbins Kaitlyn , Buchmueller Karen L 
Published: November 2023

High Mobility Group A (HMGA) proteins are a family of architectural transcription factors that control the expression of many genes. Overexpression of such proteins has been linked to metastasis in numerous cancers. HMGA proteins bind to the minor groove of DNA through two or three positively‐charged AT hook motifs, each containing the invariant sequence Arg‐Gly‐Arg‐Pro. We endeavor to investigate the effects of changing the amino acids flanking this invariant sequence using fluorimetry, circular dichroism, and filter binding. The peptides were designed to have varying flanking sequences based on amino acid occurrences in human AT hook proteins. Binding characteristics and the dynamic structure of the amino acid sequences will be elucidated by our analysis and will inform the development of HMGA‐targeting therapeutics.Support or Funding InformationBeckman Scholars Program; Arnold and Mabel Beckman Foundation
Analysis of Complex Interactions Between the Essential Subunits, Pam16, Tim44, and Tim50, of the Hsp70‐based Mitochondrial Protein Import Machinery
Authors: Yan Nicholas Lok , Ting See‐Yeun , Schilke Brenda , Craig Elizabeth A 
Published: November 2023

Because the vast majority of mitochondrial proteins are synthesized in the cytosol, efficient protein import is essential for mitochondrial function and cell viability. The mitochondrial protein import motor, which is tethered to the matrix face of the inner membrane, plays a critical role. This motor translocates preproteins from the cytosol, across both mitochondrial membranes, into the mitochondrial matrix.This biological machine is driven by the molecular chaperone mtHsp70, which binds translocating preproteins entering the matrix in an ATP‐dependent manner. Both mtHsp70 and its co‐chaperone Pam18, which performs the critical function of stimulating Hsp70's ATPase activity, are tethered to the translocon by Tim44. While mtHsp70 directly interacts with Tim44, evidence suggests that Pam18 is tethered via Pam16, with which it forms a heterodimer. However, how Pam16 interacts with Tim44 is not well understood.This study aims to determine how Pam16 interacts with Tim44 and the functional consequences of the interaction. Our previous genetic and co‐immunoprecipitation analyses implicated distinct regions in Pam16 and Tim44, both in their N‐termini, that may be the sites of interaction. Alterations in these regions not only caused Pam16 to dissociate from the translocon, but also led to temperature‐sensitive growth defects, demonstrating the importance of this interaction. We now report site‐specific crosslinking demonstrating that Pam16 and Tim44 are in close proximity to each other. When a photoactivatable crosslinker was incorporated at several positions in these regions, Pam16 could be crosslinked to Tim44, or vice versa. Altogether, these results suggest that Pam16 directly interacts with Tim44 via these regions.In addition to crosslinking to Tim44, we found that the extreme N terminus of Pam16 also crosslinked to Tim50, an essential translocon subunit that facilitates movement of the preprotein from the translocon of the outer membrane to that of the inner membrane. Tim50 is a transmembrane protein with an essential intermembrane space domain and a matrix‐exposed region whose function is unknown. Taken together, these results support the idea of Pam16 as an essential tether in the import motor, mediating complex interactions with subunits that are necessary for mitochondrial protein import.
An Automated Method for the Correction of Unsubstantiated Ramachandran Outliers in Protein Structures
Authors: Smith Connor R. , Alaniz Jacob A. , West Korbin H. J. , Weiss Charles J. , Novak Walter R.P. 
Published: November 2023

Ramachandran outliers are amino acid residues with phi and psi dihedral angles that result in energetically unfavorable conformations. While some of these conformations are supported by the data and provide key information regarding protein structure and functionality, it is currently not known how many of these outliers are actually supported by the data and how many are simply errors in the structural model. Unfortunately, in the Protein Data Bank, there exists a large number of protein structures that contain an excessive number of these outliers (>0.2%). We have developed an algorithm capable of the automated correction of unsubstantiated Ramachandran outliers. This program was built with the Python programming language using the capabilities of the protein determination software PHENIX for outlier detection and refinement.Support or Funding InformationThis research is supported by the Wabash College Haines Biochemistry Fund.
Complex structure of the disulfide bond‐dimerized PDZ‐RhoGEF and CXCR2 PDZ‐binding motif: A new mode of PDZ dimerization
Authors: Spellmon Nicholas S , Holcomb Joshua , Niu Andrea , Choudhary Vishakha , Brunzelle Joseph , Li Chunying , Yang Zhe 
Published: November 2023

Eukaryotic cellular mobility is critically dependent on actin organization and remodeling that are masterfully governed through the Rho signaling pathway activated by Rho guanine exchange factors (RhoGEF). PDZ‐RhoGEF is one unique member of the RhoGEF family which possesses a PDZ domain that is known to nucleate cell surface factors to perpetuate signaling for actin organization and cell mobility. However, the role of the PDZ domain of PDZ‐RhoGEF in signaling transduction pathways for cellular mobility is still not well understood. In this study, we identified PDZ‐RhoGEF binds directly to a novel binding partner CXCR2, a G protein coupled receptor which is known to initiate the Rho signaling program for chemotaxis, haptotaxis, cellular growth and tumor invasion. In order to understand the molecular mechanism of binding, we solved the crystal structure of the chimeric complex between the PDZ domain of PDZ‐RhoGEF and the PDZ‐binding motif of CXCR2. This complex structure reveals that the CXCR2 peptide forms an antiparallel β‐sheet against PDZ‐RhoGEF with the carboxyl‐terminal leucine packaging into a deep hydrophobic pocket within the PDZ domain. Unexpectedly, we found a disulfide bond linking the PDZ domain dimer which secures a parallel binding mode of the CXCR2 ligands to the PDZ monomers. To our knowledge, this is the first PDZ dimer structure covalently linked through a disulfide bond and may be a unique feature to PDZ‐RhoGEF family. This new type of dimerization may be important for stabilizing the CXCR2 macromolecular complex and thereby provide swift signaling for cellular mobility and cancer invasion.Support or Funding InformationNational Heart, Lung, and Blood Institute
The Conformation of Apolipoprotein E4 on Discoidal and Spherical High Density Lipoproteins Using Chemical Crosslinking and Fluorescence Spectroscopy
Authors: Bala Noor , Taiwo Kehinde , Narayanaswami Vasanthy 
Published: November 2023

Human apolipoprotein E (apoE) is 34‐kDa 299 residue exchangeable apolipoprotein that plays a critical role in lipid transport and cholesterol metabolism in the plasma and brain. The APOE gene polymorphism results in three different alleles ɛ2, ɛ3, and ɛ4, which produce the common protein isoforms, apoE2, apoE3, and apoE4, respectively. Whereas apoE3 is considered to be the anti‐atherogenic protein, apoE4 is considered a risk factor for developing Alzheimer's disease. ApoE associates mainly with vLDL but is associated with HDL‐like particles in the brain. During brain cholesterol metabolism, apoE associates with HDL to form discoidal nascent HDL which are remodeled into spherical HDL by the action of lecithin cholesterol acyltransferase (LCAT). In this study, we aim to monitor the conformational changes apoE4 undergoes during LCAT‐mediated conversion of reconstituted discoidal‐HDL (rHDL) to spherical‐HDL using chemical crosslinking and N‐(1‐pyrene)maleimide as a fluorescence probe. Single cysteine mutants of apoE4 were expressed in E. coli, purified by affinity chromatography, and reconstituted with POPC or POPC/cholesterol. The Cys residues serve as the site for attachment for the fluorescent probe or crosslinker. Discoidal rHDL (apoE4/POPC) was chemically cross‐linked using a Cys‐specific crosslinker. SDS‐PAGE revealed monomeric and dimeric bands in variants with Cys at positions 29 and 256. The dimers suggest that the Cys residues on the apoE4 mutant molecules were at a crosslinkable distance when bound to rHDL. Other residues along the length of apoE4 were not at a crosslinkable distance when bound to rHDL. To obtain spherical rHDL, recombinant human LCAT was expressed using Chinese hamster ovary cells and its specific activity was determined to be 0.8 nmol/h/mg protein using POPC/apoAI as substrate. We are currently in the process of confirming the conformation of apoE4 around the discoidal and spherical particles by an independent approach using spatially–sensitive fluorescence probes that allow us to measure the proximity of two Cys residues on two different apoE4 molecules on rHDL. It is anticipated that the combination of cross‐linker distance constraints and the excimer fluorescence emission spectra of the seven apoE4 mutants on HDL would yield an understanding of the organization of apoE4 on these particles. The significance of this study is that it offers an innovative approach to obtain insight into the structure and organization of apoE on large lipoprotein complexes. Further, it allows us to identify potential differences between apoE3 and apoE4 from a structural perspective and determine distinguishing features that contribute to the role of apoE4 in developing AD.Support or Funding InformationThis project is supported by a grant from the National Institutes of Health #GM105561.
Conformational Changes in Palladin Actin‐Binding Domains Measured by Fluorescent Resonance Energy Transfer
Authors: Womack Samuel , Vattepu Ravi , Beck Moriah Rene 
Published: November 2023

The fundamental goal of our research is to understand how cell motility in both normal and metastatic cells is regulated by palladin, an actin binding protein that plays a key role in remodeling the actin cytoskeleton. Our recent work has established that palladin regulates actin dynamics and organization by enhancing the polymerization rate and stabilizing filaments in an orthogonal meshwork. Palladin family members all utilize immunoglobulin‐like domains to bind and crosslink actin filaments. Various isoforms of palladin contain from three to five immunoglobulin‐like domains that have been implicated in a variety of protein‐protein interactions. Our previous results indicate that the Ig3 domain of palladin is directly involved in binding and crosslinking of actin and also promotes actin polymerization, whereas the Ig4 domain of palladin does not interact with actin directly. Nevertheless a tandem Ig3–4 domain protein exhibits dramatically more efficient actin binding, crosslinking, and polymerization than the isolated Ig3 domain. Recently we established that the Ig3–4 domains of palladin undergo actin‐induced dimerization, which likely results in a conformational change. NMR chemical shift measurements indicate that interactions between the tethered Ig3 and Ig4 domains are limited to the interface and lead to no more than marginal structural changes. To further narrow the gap in our understanding of palladin's role in actin assembly, we need to examine these interactions in the cellular context and demonstrate that palladin can bind directly to monomeric actin. Our current efforts are aimed at combining fluorescence resonance energy transfer (FRET) and confocal microscopy to analyze the interactions of palladin and G‐actin within cells and with isolated proteins. These experiments will also enable us to investigate the dynamic intra‐ and intermolecular interactions through the use of sophisticated fluorescent probes. Our first goal was to use site‐directed mutagenesis to replace six cysteines with alanine in two tandem immunoglobulin‐like domains of palladin before confirming that these mutations do not interfere with actin binding and bundling functions of palladin. We will share our results that make use of a newly synthesized Pacific Blue fluorescent trivalent nickel complex that we attach to the C‐terminal His6‐purification tag on our palladin constructs. We have also introduced a unique cysteine residue at the N‐terminus of palladin to allow conjugation to fluorescein maleimide so that we can track conformational changes that occur in the tandem actin‐crosslinking domains of palladin using FRET.Support or Funding InformationThis project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418 and P20 GM103638 and an undergraduate research grant from Wichita State University.
Identifying and Ameliorating Complex Collagen Misfolding Defects
Authors: Shoulders Matthew D 
Published: November 2023

Collagen is the most abundant protein in our bodies, constituting roughly one‐third of the proteome. In keeping with collagen's status as the molecular scaffold for human life, the maintenance of collagen proteostasis is one of the most critical roles of the endoplasmic reticulum (ER) proteostasis network, which is responsible for folding, modifying, processing, and quality controlling nascent collagen proteins. Dysregulated collagen proteostasis leads to numerous currently incurable diseases, including osteogenesis imperfecta. Here, we use a mechanistic biochemistry approach to decipher a cysteine‐based code for collagen heterotrimer assembly in the ER, a process that is critical for the maintenance of bone and tissue health. Moreover, we show that pharmacologic manipulation of the unfolded protein response, which maintains ER proteostasis, is able to resolve collagen folding and secretion defects that underpin osteogenesis imperfecta. These studies provide new insights into how cells solve the collagen proteostasis problem, with potentially broad implications for complex protein folding in the ER and for efforts to modulate the proteostasis network to therapeutically address the collagenopathies.Support or Funding InformationNIH NIAMS 1R03AR067503
Protein Aggregation in Ehrlichia chaffeensis during Infection of Mammalian Cells
Authors: Zolkiewski Michal , Kuczynska‐Wisnik Dorota , Cheng Chuanmin , Ganta Roman R. 
Published: November 2023

Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected ticks to humans and several other mammals. Human monocytic ehrlichiosis (HME) caused by E. chaffeensis occurs mainly in the U.S.A, but also has been reported in other parts of the world. The severity of HME varies from asymptomatic to fatal. To establish infections in the host cells, E. chaffeensis needs the capability to evade the host defenses, but it is still not clear how the pathogen responds to and overcomes the host‐induced stress. We have previously shown that the expression of the molecular chaperone clpB in E. chaffeensis is strongly upregulated immediately after infection of mammalian cells. ClpB is an ATP‐dependent heat‐shock protein with a unique disaggregase activity that reactivates aggregated proteins under cellular stress conditions. Induction of clpB in E. chaffeensis during its replication in mammalian cells suggests that the ClpB activity supports the pathogen survival inside the host, but no link between protein aggregation and a pathogen life cycle has been established so far. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the mammalian cell environment induced misfolding and aggregation of a significant fraction of E. chaffeensis proteins. The size of the aggregated fraction of E. chaffeensis proteins increased during the first 48 h post infection. The 48‐h post infection window corresponds to the upregulated production of the clpB mRNA. To disrupt the activity of ClpB in E. chaffeensis, we incubated the infected cells with up to 0.5 mM guanidinium chloride (GuHCl), which inhibits the ClpB ATPase activity in vitro. 0.5 mM GuHCl had no impact on the host cells, whereas the viability of the pathogen was reduced by ~80% in the presence of the inhibitor. Importantly, we found that the size of the aggregated protein fraction in E. chaffeensis increased significantly in cultures supplemented with 0.5 mM GuHCl. We also found that ClpB accumulated with protein aggregates formed in E. chaffeensis in the presence of GuHCl. Thus, we discovered that an exposure of E. chaffeensis to the stressful host environment during infection results in aggregation of the pathogen's proteins. Moreover, we showed that the molecular chaperone ClpB supports the survival of E. chaffeensis during the infectious stage by reducing the amount of protein aggregates. Altogether, our studies establish a novel link between the protein aggregation control and the pathogen survival.Support or Funding InformationThis project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (grant P20 GM103418), the National Institute of Allergy and Infectious Diseases (grant R01 AI070908 to RG and R56 AI121366 to MZ), and the Center of Excellence for Vector‐Borne Diseases at Kansas State University.
Divergent Mechanisms of Allosteric Regulation of Pyruvate Carboxylase by Acetyl Coenzyme A
Authors: Liu Yumeng , St.Maurice Martin 
Published: November 2023

Pyruvate carboxylase is a biotin‐dependent enzyme, composed of multiple functional domains on a single polypeptide chain, including the biotin carboxylase domain, the carboxyltransferase domain, the biotin carboxyl carrier protein domain and the allosteric domain. Acetyl coenzyme A is an allosteric activator of pyruvate carboxylase from a wide variety of species. However, the sensitivity to acetyl coenzyme A activation varies depending on the organism from which the enzyme was cloned or isolated. We are investigating the divergent allosteric activation of pyruvate carboxylase from two organisms, Rhizobium etli (RePC) and Aspergillus nidulans (AnPC). The maximum activity of RePC is accelerated ~10‐fold by acetyl coenzyme A, while AnPC is insensitive to acetyl coenzyme A activation, despite binding acetyl coenzyme A. Previous studies have structurally and kinetically characterized RePC in the presence and absence of acetyl coenzyme A. In order to investigate the divergent mechanisms of allosteric activation in AnPC and RePC, we have structurally and kinetically characterized the contributions of acetyl coenzyme A to catalytic turnover in AnPC. We have observed a highly divergent response of AnPC and RePC to acetyl coenzyme A activation in both the overall pyruvate carboxylation reaction and in the half‐reactions catalyzed at the individual catalytic domains. The binding of acetyl coenzyme A to RePC at the allosteric domain is primarily mediated by two conserved arginine residues. We generated mutations at the equivalent arginine residues in AnPC and determined that the Ka for acetyl coenzyme A was significantly increased in both mutations, indicating that acetyl coenzyme A most likely binds at the same binding site in AnPC as it does in RePC. To further clarify the mechanism of acetyl coenzyme A activation in pyruvate carboxylase, we investigated the catalytic turnover of hybrid tetramers composed of several combinations of inactivating mutations. Our data reveals that, in RePC, acetyl coenzyme A constrains the translocation of the biotin carboxyl carrier protein domain, limiting it to sample only two productive motions during catalytic turnover. However, in AnPC, acetyl coenzyme A does not alter the range of motions sampled by the biotin carboxyl carrier protein domain during catalysis. Thus, while acetyl coenzyme A binds at the same binding site in both AnPC and RePC, the divergent response of AnPC and RePC to acetyl coenzyme A activation arises, in part, from different innate carrier domain movements during catalytic turnover.Support or Funding InformationThis work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM117540‐01