bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Biochemical Characterization of a Periplasmic Lipoprotein with a Role in Adaptation of Salmonella to Magnesium Limitation
Authors: May John F , Rasmussen Damien M , Davie Timothy J , Vaneerd Cody , Groisman Eduardo A , Bhattacharyya Basudeb 
Published: November 2023

Living cells require magnesium ions for many fundamental biochemical processes. Certain pathogenic bacteria can thrive in environments with limiting magnesium, and the ability to adapt to magnesium limitation can be critical for bacterial virulence. We have used genetic and biochemical approaches to gain insight into how Salmonella enterica, a major bacterial cause of food‐borne illness, adapts to low magnesium. Using a genetic screen in a Salmonella mutant defective for adaptation to low magnesium, we identified a periplasmic lipoprotein protein that promotes growth in low magnesium. This Salmonella protein, which has a monomeric molecular weight of 19.7 kDa, contains a periplasmic localization sequence followed by a single domain of unknown function that is conserved among enterobacteria. To elucidate its function, we have purified and crystallized this protein as well as obtained atomic‐resolution X‐ray diffraction data. Circular dichroism spectroscopy indicates that the protein consists predominantly of beta‐sheet secondary structure, and the diffraction data indicate there is likely more than one protein monomer in the asymmetric unit. Overall, our results demonstrate a physiological function in magnesium ion homeostasis for a previously uncharacterized periplasmic lipoprotein in Salmonella, and we have obtained the first atomic‐resolution structural data for a member of this family of proteins. These discoveries will provide insight into molecular mechanisms of bacterial adaptation to magnesium limitation.Support or Funding InformationThis research is supported by University of Wisconsin‐La Crosse Undergraduate Research and Creativity Grants (DR, TD), the McNair Scholars Program at UW‐La Crosse (DR), and a University of Wisconsin‐La Crosse Faculty Research Grant (JM). The authors would also like to thank Professor James Keck (University Wisconsin, Department of Biomolecular Chemistry) for graciously allowing access to the Advanced Photon Source. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006).
Characterization of the Role for the N‐Terminal Domain on Sil1 Activities as a Nucleotide Exchange Factor and Reductase
Authors: Pareja Kristeen Alcaide , Sevier Carolyn 
Published: November 2023

The accumulation of reactive oxygen species (ROS) can lead to oxidative stress and play a role in human diseases such as cancer and neurodegenerative diseases. We are interested in studying how cells use ROS as signaling molecules to maintain a balanced redox environment. Using yeast as our model system, we have previously shown that the Hsp70 in the endoplasmic reticulum (ER), BiP, acts as a direct sensor of peroxide, a type of ROS produced during protein folding. BiP has two major domains that regulate its function as a chaperone: an ATPase domain and a substrate‐binding domain. Under ER oxidative stress, yeast BiP's conserved cysteine gets modified/oxidized by peroxide. Modification of BiP's cysteine causes a conformational change, which enhances BiP's ability to bind to substrates, leading to protection of cells under ER oxidative stress. When cells go back to non‐stress conditions, reversibility of BiP's cysteine modification is important for normal cellular function and survival. We have evidence that the protein Sil1, a known nucleotide exchange factor (NEF) of BiP, can remove the modification on BiP's cysteine. We have shown that in addition to acting as a NEF, Sil1 has an unexpected reductase activity that depends on two cysteines, Cys‐52 and Cys‐57, located at the N terminus. While the N‐terminal domain is required for Sil1 activity as a reductase, Sil1 is competent as a NEF even in the absence of the N‐terminal domain. Our goal is to further elucidate the molecular details relating to how the N‐terminal region of Sil1 impacts both Sil1 NEF and reductase activity by utilizing a variety of biochemical and biophysical approaches. Our preliminary data suggest that NEF activity is influenced by the presence of the N terminus. Our current data also suggest the N‐terminal domain may adopt multiple conformations, allowing for regulation of Sil1 NEF activity and potentially its activity as a reductase. Of note, mutations in the Sil1 gene have been found in over half of patients with Marinesco‐Sjögren Syndrome (MSS), a rare autosomal disease characterized by early onset cataracts, cerebellar ataxia, and delayed mental development. One such mutations maps near the N‐terminal domain, and we expect that characterization of full‐length Sil1 may also lend further insight into how mutations in Sil1 can cause MSS.Support or Funding InformationNSF GRFP and SUNY Diversity Fellowship
An Alternative Structural Model of Activation for the Anti‐Anti‐σ Factor PhyR and Interaction with the Anti‐σ Factor NepR
Authors: Luebke Justin L , Eaton Daniel S , Sachleben Joseph R , Crosson Sean 
Published: November 2023

Protein phosphorylation is a universally conserved post‐translational modification. In bacteria, aspartyl phosphorylation generally controls transcriptional regulatory proteins, enabling cells to respond to chemical and physical changes in their local environment. In the large and diverse alphaproteobacteria class of bacteria, stress monitoring is primarily regulated by the general stress response (GSR). PhyR is the master regulator of the GSR and is a two‐domain anti‐anti‐σ factor containing a phosphorylatable response regulator receiver domain and a σ‐like output domain. PhyR titrates the anti‐σ factor NepR away from its cognate σ factor in a phosphorylation‐dependent manner to ultimately control global changes in gene expression. The binding site of NepR within PhyR is largely occluded at the domain interface. It has long been hypothesized that phosphorylation of PhyR results in undocking of the two domains to expose the high‐affinity NepR binding site; however, the foundations by which phosphorylation of the PhyR receiver domain controls structure and function remains largely undefined. Small‐angle X‐ray scattering (SAXS) and molecular dynamics (MD) simulations indicate phosphorylation is insufficient to drive PhyR opening. Recent biophysical and biomolecular NMR data indicate intrinsically disordered regions of NepR play a significant role in PhyR activation. These interactions drive an unstructured to structured transition of NepR and are required to facilitate both PhyR phosphorylation and PhyR‐NepR binding. This leads to a model where phosphorylation is necessary but insufficient to expose the high affinity NepR binding site of PhyR and interactions between PhyR and NepR prior to phosphorylation are a necessary component of the structure‐function relationship of these two core regulatory proteins.Support or Funding InformationNIH 5R01AI1707159
Characterization of the Ordered Domain of an Epstein Barr Viral Tegument Protein
Authors: Marlowe Megan E. , Gorres Kelly 
Published: November 2023

Epstein Barr virus (EBV) is the first known human herpes virus to cause cancer. The virus also causes mononucleosis. Within the viral life cycle, the virus can switch between latent and lytic phases. In the latent phase, very few genes are expressed and new virions are not produced. During the lytic, or active, phase there is an increase in viral gene expression, proteins are produced, and infectious viral particles are released from the cell. The structure of Epstein‐Barr virus, like all herpesviruses, contains a tegument layer, which surrounds the genome‐containing capsid. The proteins in the tegument are delivered to the cell immediately upon infection. These proteins conduct a wide variety of functions in the viral life cycle. This project investigates an EBV tegument protein essential for virus production, but its role is unknown. Bioinformatics analysis identified homologs in other herpesviruses, but no similarity to any protein in any other organism. Amino acid sequences within a protein can exhibit order and disorder, relative to its three dimensional structure. A database called MobiDB was used to predict regions of order versus disorder from the amino acid sequence. The C‐terminal portion of the protein is calculated to be low complexity or disordered, whereas an N‐terminal domain is ordered. To express this structured part of the protein, an early stop codon was inserted into the gene. The protein with a His‐tag was overexpressed in E. coli and purified by nickel‐affinity chromatography. The objective of the experiments conducted is to study the function and characteristics of the structured domain of a tegument protein expressed in the lytic phase of the EBV infection.Support or Funding InformationThis work was supported by the UWL College of Science and Health and a UWL Faculty Research Grant to K.G.
Structure, Dynamics and Folding of an Immunoglobulin‐like Domain of Actin Binding Protein Palladin
Authors: Vattepu Ravi , Beck Moriah R 
Published: November 2023

While members of the immunoglobulin (Ig) share a very conserved folding pattern, this family is also the most diverse in terms of function and sequence. The conserved ®–sandwich fold of Ig domains provides a stable scaffold to display a variety of binding site sequences on its surface, however it has been suggested that small variations in the fold may add additional flexibility to fulfill specific functions. Among the few known intracellular proteins containing Ig‐domains, the muscle‐related family is the most common and includes cell adhesion molecules like titin and telokin. Palladin is a recently identified protein from a novel family of actin binding protein that includes myopalladin and myotilin. All of these family members utilize immunoglobulin‐like (Ig) domains to bind and crosslink actin filaments. Palladin isoforms contain three to five immunoglobulin‐like (Ig) domains that have been implicated in a variety of protein‐protein interactions. Our previous results suggest that palladin Ig3 domain is directly involved in binding and crosslinking of actin, promoting actin polymerization, and phospholipid binding. Although the Ig4 domain of palladin does not interact with actin, the tandem Ig3–4 domains display much more efficient actin binding, crosslinking, lipid binding and polymerization than Ig3 alone. Recently we established that palladin dimerizes upon binding to actin, which likely results in a conformational change. Now we have carried out a detailed structural and dynamical characterization of the isolated Ig3 and Ig4 domains of palladin by NMR spectroscopy to provide a basis for understanding the underlying differences between the isolated and tandem domain functions. In the present study, we describe the use of a range of solution‐state NMR techniques and other biophysical methods to monitor dynamics and folding properties of the Ig3 and Ig4 domains of palladin. We have found that the most flexible regions of the Ig3 domain include regions that overlap with actin binding sites, suggesting that dynamics may be critical for function. We also employ NMR backbone relaxation measurements to explore conformational exchange taking place at the hydrophobic core of both Ig domains and compare these results to other closely related proteins.Support or Funding InformationThis project was supported by an Academic Research Enhancement Award (AREA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number R15 GM120670 and start‐up funds from Wichita State University.
Salt‐Dependent Protein Splicing in Extreme Halophiles
Authors: Janton Christopher J. , Reitter Julie N. , Mills Kenneth V. 
Published: November 2023

Protein splicing is an autocatalytic, post‐translational process by which an intervening polypeptide, or intein, can excise itself and ligate the flanking polypeptides, or exteins. Inteins have been found in a species of extreme halophilic archaea, Halobacterium salinarum (Hsa). Previous work in the lab demonstrated that splicing of the Hsa DNA Polymerase II (Polll) intein is salt‐dependent. We compared the in vitro activity of the Hsa Polll intein to that of the only other Hsa intein, which interrupts the Cell Division Control protein (Cdc21a). The Hsa Cdc21a intein does not splice in E. coli, but can be induced to splice at very high salt concentrations under reducing conditions in vitro. Additionally, we investigated the salt dependence of isolated C‐terminal cleavage of the Hsa Polll splicing mechanism. We plan to test the splicing activity of the Hsa Cdc21a intein in vivo.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).
Modeling Dynamics in the D‐Amino Acid Oxidase Protein
Authors: Kueffer Lauren , Beyers Wyatt 
Published: November 2023

In human brains the enzyme D‐amino acid oxidase (hDAAO) plays a key role in modulating the degradation pathway of the important signaling molecule D‐serine. The hDAAO enzyme is active as a homodimer and is stereospecific for the oxidative deamination of D‐amino acids to give ammonia and an α‐keto acid, coupled to the reduction of the cofactor FAD. The reoxidation of FAD results in hydrogen peroxide. The deregulation of D‐serine signaling has been linked to schizophrenia susceptibility and certain mutations in the hDAAO enzyme itself are associated with familial amyotrophic lateral sclerosis (ALS). Mutations in the hDAAO enzyme are known to impact enzyme function and stability.We were interested in comparing apo‐enzyme simulations to FAD bound simulations of the wild‐type hDAAO enzyme, a wild‐type pig kidney D‐amino acid oxidase enzyme (pkDAAO), a mutant R279A hDAAO enzyme, and a mutant D31H hDAAO enzyme. Even though there is 85% sequence identity between the two species, hDAAO binds FAD weaker and shows a slower rate of flavin reduction compared to pkDAAO. The R279A mutation exchanges an Arginine for an Alanine at position 279 on both chains of the homodimer. This mutation is close to the FAD binding site and has shown to increase the enzyme's binding affinity for FAD. The D31H mutation exchanges an Aspartate for a Histidine at position 31 on both chains of the homodimer. Even though residue 31 is located far from the FAD binding site, this mutation leads to a higher binding affinity for FAD compared to the human wild‐type. We use molecular dynamic simulations to study how the protein structure changes over time and how the protein dynamics are altered by mutations in the amino acid sequence. Investigating the changes we see at the atomic level in protein structure can help to determine how each mutation can have different effects on enzyme stability or activity.
Propagation of tau prions from Alzheimer's disease and chronic traumatic encephalopathy patients in cultured cells
Authors: Woerman Amanda L , Aoyagi Atsushi , Patel Smita , Kazmi Sabeen A , Lobach Iryna , Grinberg Lea T , McKee Ann C , Seeley William W , Olson Steven H , Prusiner Stanley B 
Published: November 2023

Tau prions aggregate in the central nervous system, giving rise to neurodegeneration. Among the tauopathies, Alzheimer's disease (AD) is the most common, whereas chronic traumatic encephalopathy (CTE), Pick's disease (PiD), and progressive supranuclear palsy (PSP) are less prevalent. Brain extracts from deceased individuals with PiD, a neurodegenerative disorder characterized by three‐repeat (3R) tau prions, were used to infect HEK293T cells expressing 3R tau fused to yellow fluorescent protein (YFP). Extracts from argyrophilic grain disease (AGD), corticobasal degeneration (CBD), and PSP patient samples, which contain four‐repeat (4R) tau prions, were transmitted to HEK293 cells expressing 4R tau fused to YFP. These studies demonstrate that prion propagation in HEK cells requires isoform pairing between the infecting prion and the recipient substrate. Interestingly, tau aggregates in AD and CTE, which contain both the 3R and 4R tau isoforms, were unable to robustly infect either 3R‐ or 4R‐expressing cells. However, AD and CTE prions were able to replicate in HEK293T cells expressing both 3R and 4R tau. Unexpectedly, increasing the level of 4R isoform expression alone supported the propagation of both AD and CTE prions. The finding that tau prion strains are isoform specific may have important implications for developing anti‐tau therapeutics and disease‐specific PET probes.Support or Funding InformationThis work was supported by NIH Grants AG002132 and AG031220, Daiichi Sankyo, Dana Foundation, Glenn Foundation, Sherman Fairchild Foundation, and a gift from the Rainwater Charitable Foundation. Control, AD, AGD, CBD, PiD, and PSP tissue samples were provided by the University of California, San Francisco, Neurodegenerative Disease Brain Bank, which is supported by NIH Grants AG023501 and AG19724 (to W.W.S.), Tau Consortium, and Consortium for Frontotemporal Dementia Research. CTE tissue samples were provided by the Boston University Alzheimer's Disease Center Chronic Traumatic Encephalopathy Center, which is supported by the Department of Veterans Affairs, National Institute of Neurological Disorders and Stroke, National Institute of Biomedical Imaging and Bioengineering (NS086559), National Institute on Aging, Boston University Alzheimer's Disease Center (AG13846; Supplement 0572063345–5), Concussion Legacy Foundation, Andlinger Foundation, and World Wrestling Entertainment, Inc.
Protein Aggregation Small Molecule Inhibitor Discovery and Mechanisms
Authors: Xu Bin 
Published: November 2023

Epidemiological and clinical studies showed significant association between type 2 diabetes (T2D) and the risk for Alzheimer's disease. Pancreatic hormone amylin is a highly amyloidogenic protein and amylin amyloid deposition in the pancreas are hallmark features of T2D. Recent clinical studies showed that amylin plaques were deposited in the brain of diabetic patients, but not in healthy controls. We performed cell‐based studies, demonstrating that amylin amyloid is highly toxic to pancreatic β‐cells INS1 and neuronal cells SH‐SY5Y and Neuro2A. From a collection of natural compounds used in alternative medicine, we identified multiple potent inhibitors, including rosmarinic acid (RA) and baicalein (200 nM and 1 μM respectively in apparent IC50). These lead compounds disaggregate amylin fibrils from transmission electron microscopic observations and significantly reduce amylin‐induced cytotoxicity. Dissecting the functional groups of these compounds, we demonstrated, for the first time to our knowledge, that the vicinal hydroxyl groups of the catechol groups played key functional roles in amyloid inhibition in more than a dozen catechol‐containing compounds, including physiological neurotransmitters epinephrine, norepinephrine, and dopamine. Compounds with multiple catechol groups, such as rosmarinic acid, exhibited additive/synergistic effects. We provided further mass spectrometric evidence that incubating several of these catechol‐containing inhibitors with amylin leads to covalent adducts consistent with Schiff base conjugation as a mechanism for blocking toxic amyloid formation. The inhibition effects by these compounds were also demonstrated in molecular simulation analyses, providing additional non‐covalent inhibition mechanisms. To expand protein aggregation inhibitor discovery, we applied the concept of drug repurposing. We developed a 384‐well plate based screening platform and screened a NIH Clinical Collection small molecule library that are currently in phased trials. We were able to rapidly identify 10–30 each of “hits” against four different amyloidogenic proteins: amylin, Aβ, tau, and α‐synuclein. Excitingly, we were able to identify compounds that can inhibit all four amyloids but also “protein‐specific” inhibitors. Mechanisms for “general” protein aggregation inhibitors and for “protein‐specific” aggregation inhibitors will be elucidated.Support or Funding InformationThis work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, Diabetes Action Research and Education Foundation (DAREF), and Virginia Tech Center for Drug Discovery (VTCDD).
Characterization of Anti‐SOD1 Antibodies and Detection of Intermediary SOD1 Oligomers
Authors: Atlasi Ryan Saleh , Malik Ravinder , Corrales Christian , Tzeplaeff Laura , Cashman Neil , Bitan Gal 
Published: November 2023

Mutations in the gene encoding the enzyme copper‐zinc superoxide dismutase (SOD1) are linked to 10–20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations lead to misfolding and self‐assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. However, the molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization and detection of SOD1 oligomers is particularly challenging due to their metastable nature. Antibodies against oligomeric and misfolded SOD1 forms are useful tools for this purpose, provided their specificity and selectivity are well characterized, which is not always the case. In this study, we set out to characterize three novel anti‐misfolded SOD1 antibodies and compare them with two commercial anti‐misfolded SOD1 antibodies raised against G93A SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti‐SOD1 antibodies that were expected to be insensitive to misfolding. We asked whether the antibodies could distinguish among native and misfolded conformations of SOD1 and whether any of them were specific for the elusive oligomeric forms of the protein. To follow SOD1 conformational change and aggregation kinetics, wild‐type and G93A SOD1 were incubated under reducing conditions and the reactions were followed using thioflavin T (ThT) fluorescence, electron microscopy (EM), and dot blots with each antibody. Control reactions were followed under non‐reducing conditions. The three new anti‐misfolded SOD1 and one of the commercial antibodies (B8H10) showed maximum affinity at the time oligomers were expected to dominate and lost affinity with fibril formation. Unexpectedly, the two polyclonal antibodies also showed preference for oligomers, especially of G93A SOD1. Surprisingly, the other commercial anti‐misfolded SOD1 antibody, C4F6, showed specificity for G93A SOD1 regardless of misfolding. Two of the new antibodies showed preference for early or late oligomers, suggesting that they could expand currently detectable temporal resolution. In conclusion, our data indicate that certain antibodies are highly sensitive to the misfolded form of SOD1, whereas others are insensitive to either mutation or aggregation.Support or Funding InformationRGK Foundation