bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Role of the N‐terminal Region of bHLH TWIST Proteins in the DNA‐binding Activity of ADD1/SREBP1c
Authors: Rodriguez Yacidzohara , Cadilla Carmen L 
Published: November 2023

TWIST1 and TWIST2 proteins are key bHLH transcription factors involved in the inhibition of differentiation of mesenchymal cell lineages during early stages of embryogenesis. Besides using the common bHLH domain for protein‐protein interactions with other proteins, they are also able to use their N‐ and C‐terminus domains. Although the transcriptional regulation of interacting proteins by use of their bHLH and C‐terminus has been extensively studied, there is little information that directly compares the N‐terminus of both Twist proteins interacting with a common partner. ADD1/SREBP1c is a transcription activator whose activity has been shown to be repressed by TWIST2, mainly by reducing its DNA binding to target sequences. To determine whether TWIST2 and TWIST1 may employ their N‐terminal regions to interact with ADD1, we used truncated TWIST2 (Q65X) and TWIST1 (Q107X) proteins containing only their N‐terminal regions. EMSA analysis showed reduced DNA binding of ADD1 homodimers when co‐expressed with Q65X, which suggests that TWIST2 uses its N‐terminus to interact with ADD1. In contrast, the DNA binding of ADD1 was not affected when co‐expressed with mutant TWIST1 Q107X, suggesting that the N‐terminus of TWIST1 does not interact with ADD1. To determine the region of TWIST2 responsible for the interaction with ADD1, we performed deletion mutagenesis of two conserved sub‐motifs found in the N‐terminus: SSSPVSP (Twist2ΔA) and SEEE (Twist2ΔB). EMSA analysis showed ADD1 DNA binding as homodimers when co‐expressed with Twist2ΔB but not when Twist2ΔA was co‐expressed, suggesting that TWIST2 uses the SEEE sub‐motif to interact with ADD1. To determine whether the sequence differences in the N‐terminal region of TWIST2 and TWIST1 account for the functional difference in protein‐protein interactions with ADD1, deletion mutagenesis of TWIST1's glycine‐rich regions was conducted. EMSA results showed that like WT TWIST2, removal of TWIST1's both glycine‐rich regions allows interaction between TWIST1 and ADD1, as seen by the loss of ADD1's binding to the DNA. To determine which of the two glycine‐rich regions is used for the interaction with ADD1, we deleted either the first glycine‐rich region (Twist1ΔGly1) or second glycine‐rich region (Twist1ΔGly2). EMSA results demonstrate that when ADD1 is co‐expressed with Twist1ΔGly1, the DNA binding activity of ADD1 is greatly increased. However, when co‐expressed with Twist1ΔGly2, DNA binding activity of ADD1 is reduced. This suggests that the first glycine‐rich region of TWIST1 could be responsible for the interaction with ADD1. In conclusion, TWIST1 and TWIST2 proteins use different regions of their Amino‐terminus to differentially regulate the DNA‐binding activity of ADD1/SREBP1c.Support or Funding InformationResearch was supported by grants: G12MD007600 (RCMI) and R25GM061838 (RISE)
Investigating the Role of a Dual Motif in DNA Binding by PAX3‐FOXO1
Authors: Weatherspoon Amber , Evans Ronise , Cherlin Tess , Bratton Melyssa , Johanson Kelly 
Published: November 2023

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma that can be divided into two main categories. Embryonal Rhabdomyosarcoma (ERMS) usually affects children in the first five years of life and has not been linked to the presence of specific gene fusions. Alveolar Rhabdomyosarcoma (ARMS), which is more aggressive, typically affects all age groups equally but a larger portion of RMS occur in older children. The more aggressive form of ARMS is primarily characterized by a reciprocal (2;13) (q35; q14) translocation, which results in the fusion of the N‐terminus of PAX3 and the C‐terminus of FOXO1. PAX3‐FOXO1 mediated gene activation leads to ARMS development has been linked to several events involved in ARMS development, although all targets of this fusion protein have not been identified. A search of an updated ChIP‐seq screen of 3881 PAX3‐FOXO1 putative binding sites revealed that 36% contained both a PAX3 paired domain and FOXO1 recognition sequence within proximity. In vitro examination of a PAX3‐FOXO1 dual motif indicates that mutation of the FOXO1 site or residues in the FOXO1 DNA‐binding domain affect both binding and activation of these sequences. Binding sites for other transcription factors overlap or surround several of these dual motifs, indicating the need to examine potential transcriptional complexes formed by PAX3‐FOXO1. Understanding the binding specificities and activities of this fusion protein will allow for a better understanding of the transcription factor which may lead to future therapeutic remedies.Support or Funding InformationNIH BUILD RL5GM118966‐02, NIH RCMI 2G12MD007595‐06, NIH BUILD TL4GM118968
Protein‐Protein Interactions in Nickel Acquisition of Escherichia coli
Authors: Zeng Zhi Wei , Zamble Deborah 
Published: November 2023

Nickel is a required cofactor for [NiFe]‐hydrogenase, an enzyme involved in bacterial metabolism and pathogenesis.1 Due to the toxicity of nickel,2 bacteria need to control its uptake and distribution intracellularly.3 In Escherichia coli, control of intracellular nickel is achieved by nickel uptake by membrane transporters and nickel delivery to the correct destinations by metallochaperone proteins; however, it is not clear how these two systems are linked. Previous results suggested that there is an interaction between the nickel metallochaperone HypB and a nucleotide binding domain of Nik transporter NikE. This protein‐protein interaction would support the hypothesis that HypB, a key protein in nickel delivery to the [NiFe]‐hydrogenase precursor protein, receives nickel directly from the Nik transporter. In the present work, the NikE‐HypB interaction is examined under various nucleotide‐loaded and metal‐loaded states of HypB.Support or Funding InformationThis work was supported in part by funding from NSERC and CIHR.
Secondary Structure Analysis of the C‐terminus of Ga‐interacting Vesicle Associated Protein Using Circular Dichroism Spectroscopy
Authors: Maddox Adam L , Bhandari Deepali 
Published: November 2023

A vast majority of cell signaling is mediated through activation of hetero‐trimeric G proteins. Ga‐Interacting Vesicle associated protein (GIV) is a non‐receptor Guanine nucleotide exchange factor (GEF), which activates Gi family of heterotrimeric G proteins downstream of activated receptor tyrosine kinases (RTKs). GIV's GEF function is mediated by a stretch of highly conserved ~20 residues, which is followed by a putative SH2‐like domain in the C‐terminus of the protein. Previous studies have shown that the C‐terminal 211 amino acids of GIV (referred to as “GIV‐CT” henceforth) are capable of functioning autonomously from its recruitment to the cell surface upon activation of RTKs to promoting downstream signals by binding to and activating Gi proteins. However, despite all the functional information and computational predictions, the structural insights into how GIV‐CT is able to perform all these functions is missing. Here, we have attempted to probe the structural aspects of GIV‐CT using circular dichroism (CD) spectroscopy ‐ the most commonly used method for determining the secondary structure of peptides and proteins. N‐terminally His6‐tagged GIV CT wild type (WT) and a phosphomimetic mutant (S1674D; binds and activates Gai better than the WT) were expressed and purified from E. coli BL21‐DE3 cells using Co2+‐NTA affinity chromatography followed by cation exchange chromatography. Our preliminary CD spectroscopy analyses showed a random coil profile for GIV CT WT as well as S1674D mutant, both of which could be induced to adopt an a‐helical conformation by addition of trifluoroethanol (TFE). Further analysis of CD spectra to predict secondary structure characteristics was carried out using DichroWeb, an online deconvolution program, which compares CD data for known protein structure to that of unknown protein structure. Wild type GIV CT secondary structure was predicted to contain 1% α‐helix, 18% β‐sheet and 46% random coil while our mutant contained 1% α‐helix, 15% β‐sheet and 56% random coil. B‐turn and ‘denatured’ helix and sheet percentages make up the final total of 100% secondary structure. In the presence of TFE, GIV CT WT and 74D α‐helical content increased to 24% and 28%, respectively. Together, our data suggest that although recombinant GIV‐CT may be predominantly in an unstructured state, it likely has a propensity to fold into a regular secondary structure, perhaps upon interacting with a binding partner.Support or Funding InformationThis project is supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638.
Evaluating the role of a multi‐heme cytochrome c in electron transfer from an electrode surface to Heliobacterium modesticaldum
Authors: Herrera‐Theut Kathryn Ann , Gisriel Christopher , Laureanti Joseph , Orf Gregory , Baker Patricia , Jones Anne Katherine , Redding Kevin 
Published: November 2023

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can produce H2 when other electron transfer pathways are restricted. In addition, electrons can be injected into this organism via an electrode and redox mediator in a light‐dependent fashion, as shown recently by the Redding and Jones research groups. These factors make H. modesticaldum an ideal organism for use in a microbial photoelectrosynthesis cell, in which electricity can be used to power specific metabolic processes that produce a desired compound (e.g. H2). However, the injection of electrons into this organism has not yet been optimized. There is a gene (HM1_0653) in the genome encoding a multi‐heme cytochrome c that is similar to the proteins known to be used for exit of electrons in the well‐known electrode‐respiring bacteria (e.g. Geobacteria). RNA‐sequencing in the Redding lab has shown that the HM1_0653 gene is very poorly expressed in H. modesticaldum. Boosting expression of this cytochrome could lead to faster electron transfer into the cells and thereby more H2 production. This hypothesis is being tested by cloning the HM1_0653 gene into a plasmid behind a strong promoter and introducing it into H. modesticaldum using a transformation system recently developed in the Redding lab. To test that the protein is being highly expressed, SDS‐PAGE/heme‐staining will be conducted. The effect of HM1_0653 on electron transfer will then be assessed by comparing the light‐dependent current into cells containing the overproducing plasmid with control cells, as well as H2 production.Support or Funding InformationI would like to thank Arizona Power Authority and the Arizona State University College of Liberal Arts and Sciences Scholarship for their support.
Conformational landscape of the protein kinase A Inhibitor PKI studied by fluorescence and NMR spectroscopy
Authors: Olivieri Cristina , Li Geoffrey , Kim Jonggul , Porcelli Fernando , Neibergall Matthew , Muretta Joseph M. , Thomas David D. , Taylor Susan S. , Gianluigi Veglia 
Published: November 2023

Cyclic AMP‐Dependent protein kinase A (PKA) is a ubiquitous phosphoryl transferase that participates in numerous cellular signaling pathway. Inside the cell, the activity and localization of PKA are finely regulated by endogenous inhibitor proteins, such as the heat‐stable protein kinase A inhibitor (PKI). PKI plays a key role also in the nuclear regulation and cytoplasmic translocation of the catalytic subunit of PKA (PKA‐C). To understand the molecular mechanism underlying recognition of PKA‐C by PKI, we characterized the change in the conformational landscape of PKIα upon binding PKA‐C as well as the mechanism of binding, using solution NMR and fluorescence spectroscopy. We demonstrated that PKI free in solution has minimal secondary structural elements with only two transient helices and has transient intramolecular interaction between the C‐ and N‐terminus. When a ternary complex between PKA‐C, PKI and a non‐hydrolysable ATP mimic (ATPγN) is formed, only the portion of the peptide that directly interacts with the PKA‐C undergoes significant structural rearrangement, while the majority of the peptide is still unfolded. However, our experiments also indicated that binding to PKA‐C confers allosteric changes toward the nuclear export signal (NES) region present on PKI. Also our transient kinetics data reveal that the mechanism involves at least two distinct populations of PKA‐C binding to PKI with subsequent structural rearrangement and the flux through these competing pathways depend on ligand concentration. These studies not only elucidate the structural and mechanistic basis of PKA‐C:PKI interaction, but also represent the basis for further investigation on the role of the PKA‐C:PKI interaction toward regulation and nuclear export.Support or Funding InformationNIH GRANT R01 from Gianluigi Veglia GM100301‐05
Crystal Structures of a Bacteriophytochrome from Myxobacteria Stigmatella aurantiaca
Authors: Waltz Patricia M , Halavaty Andrei , Patel Hardik , Hopkins James , Woitowich Nicole , Stojkovic Emina 
Published: November 2023

Bacteriophytochromes (BphPs) are red‐light photoreceptors that regulate light responses in bacteria via reversible photoconversion between red (Pr) and far‐red (Pfr) light‐absorbing states. BphPs consist of a photosensory module that is composed of the PAS (Per‐ARNT‐Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains. PHY domain is covalently linked to a signaling‐effector module such as histidine kinase. Wild type protein from non‐photosynthetic myxobacterium Stigmatella aurantiaca (SaBphP1) was purified, crystallized, and the structure of the complete photosensory module was determined at 2.35 Å resolution. Unlike classical BphPs, SaBphP1 undergoes limited Pr/Pfr photoconversion in the presence of the PHY domain. Wild‐type SaBphP1 lacks a conserved His that forms a hydrogen bond with the chromophore in the Pr state in the classical BphPs. A single Thr289His mutation in the chromophore‐binding pocket of SaBphP1 results in a complete Pr/Pfr photoconversion. Consequently, we determined the crystal structure of the complete photosensory module for SaBphP1 Thr289His mutant at 2.85 Å resolution. Both SaBphP1 crystal structures, solved in the Pr state, reveal how interdomain interactions bring together PAS, GAF and PHY domains to achieve Pr/Pfr photoconversion. We identify key amino acids that stabilize the chromophore in the Pr state and provide structural evidence to support the essential role of the PHY domain in Pr/Pfr photoconversion. Our results highlight local structural changes from the photosensory domain that significantly affect the long, signaling helices at the dimer interface and may impact the spatially distant histidine kinase domain and its activity.Support or Funding InformationNSF MCB RUI 1413360
Evaluation of a Prefusion Model of the Herpesvirus Fusion Protein via Mutagenesis
Authors: Robinson Carolyn A , Gallagher Shannon L , Connolly Sarah A 
Published: November 2023

Herpesviruses affect a significant portion of the human population and cause a variety of diseases, including cold sores, chickenpox, mononucleosis, and some forms of cancer. The first step of any viral infection requires a virus to enter a host cell. Herpesviruses enter cells by fusing a lipid viral envelope with the host cell membrane. Glycoprotein B (gB), a virally‐encoded class III fusion protein, mediates this fusion event by physically inserting into the host cell membrane and refolding from a prefusion to a postfusion conformation. The crystal structure of gB in its postfusion form has been solved, but its prefusion structure is unknown. A model of the prefusion gB structure was created based on homology to another class III fusion protein. To assess this model, we introduced mutations into gB that we predicted would affect its fusion capacity by altering the stability of its prefusion conformation. Mutations were designed to increase the flexibility of a coiled‐coil that is extended in the postfusion structure but broken in the prefusion model. gB is an essential protein that is conserved in all herpesviruses. Insights into the molecular details of how the gB protein refolds to force membranes to fuse may identify targets for antiviral intervention.Support or Funding InformationDePaul University Research Council Grant
The Role of a Highly Conserved Loop in Response Regulator Activation in the General Stress Response
Authors: Eaton Daniel S , Luebke Justin L , Crosson Sean 
Published: November 2023

Bacteria depend on a diverse array of signaling proteins to sense and respond to their environments. An important class of proteins involved in such signaling cascades are the response regulators, which are controlled by the phosphorylation state of a conserved aspartate residue. Though most response regulators share great structural similarity, the allosteric pathway that governs their activation upon phosphorylation tends to vary. In this study, we examine the response regulator PhyR, a two‐domain protein comprised of a phohsphorylatable receiver domain and a σ‐like domain. This protein functions as an anti‐anti‐σ factor that governs activation of the general stress response (GSR) in alphaproteobacteria. Activated PhyR accomplishes GSR induction by binding to its cognate anti‐σ factor, NepR, through its σ‐like domain. This domain is occluded by the receiver domain in the inactive state. As such, in order for PhyR to carry out its binding activity, its receiver domain must undergo a significant conformational change to detach from the σ‐like domain. While a number of structural studies have focused on PhyR, a full characterization of the active state of the protein remains elusive. In the absence of definitive structural information about the protein's active state, we probed PhyR's activation mechanism through molecular dynamics (MD) simulations. From these simulations, we predicted that, for the protein to activate, a highly conserved loop in the receiver domain must be restructured in a manner which would disrupt a set of strong hydrogen bonds. Mutating the PhyR residues involved in these interactions results in hyperactive binding to NepR. Thus, we show that disruption of a particular interaction interface is sufficient to bias PhyR into the active state and therefore these residues play an important role in its activation dynamics.Support or Funding InformationFunding: NIH 5R01Al107159
The B domain of the mitochondrial fission mechanoenzyme Drp1 is intrinsically disordered and coacervates under crowding conditions
Authors: Hill Blake , Posey Ammon , Bagheri Mehran , Kennedy Nolan , Khan Misha , Harden James 
Published: November 2023

The Dynamin superfamily is a class of large GTPases that perform essential membrane remodeling events via self‐assembly stimulated GTP hydrolysis in a manner that involves the coordination of lipid‐membrane interaction, high‐order oligomerization, and GTP hydrolysis, however a clear understanding of how these functions are coordinated has remained elusive. A “variable domain” (VD) is present in many superfamily members and may enable specific targeting to sites of action, but the nature of VD function is unclear in most dynamins. Dynamin‐related protein 1 (Drp1) is the primary mechanoenzyme responsible for mitochondrial fission. We seek to identify the role of the Drp1 VD, or B domain. We find that removal of the B domain from Drp1 results in enhanced assembly and GTP hydrolysis, suggesting that the B domain has an auto‐inhibitory role. We find that the B domain is intrinsically disordered (ID) and surprisingly, undergoes phase separation or coacervation under conditions that induce other ID proteins to fold. The same conditions that induce phase separation of the B domain also enhance binding to cardiolipin, a lipid that is unique to the mitochondrial membrane. Based on these findings we suggest a model in which the B domain inhibit Drp1 assembly by dynamically occluding assembly interfaces, and that this auto‐inhibition is relieved upon interaction of the B domain with cardiolipin at the mitochondrial membrane, involving the process of coacervation. This model may also be applicable to other members of the dynamin superfamily, and intrinsically disordered membrane remodeling proteins in general.Support or Funding InformationNIH R01‐GM067180
Regulatory role of non‐phosphorylated tau 441 and its antibodies on tubulin polymerization
Authors: Imhof Matthew M. , Zabik Nicole L. , Martic Sanela 
Published: November 2023

Tau protein is a microtubule (MT) associated protein which functions to promote polymerization and stability of microtubules (MT) in neuronal cells. When tau undergoes post‐translational modification, it misfolds and aggregates, forming cytotoxic species which lead to neurodegeneration. Immunotherapies against tauopathies have shown promise in animal models by inducing clearance of tau pathology. However, the exact mechanisms of antibody‐based inhibition of neurodegeneration remain unidentified. We have reported on anti‐tau antibodies to non‐phosphorylated tau441 and their inhibition of tau aggregation in vitro. We also showed that phosphorylation of tau441 at Ser199, by GSK‐3β, may be inhibited by anti‐tau phosphospecific antibodies. Here, we have evaluated polymerization of tubulin in the presence of non‐phosphorylated tau441 and anti‐tau antibodies to non‐phosphorylated tau [targeting epitopes in the N‐terminal, R4, and C‐terminal]. ELISA was used to determine antigen‐antibody binding affinities between tau, tubulin and anti‐tau antibodies. An anti‐tubulin antibody was used as a control in the polymerization and ELISA experiments. Using the tubulin fluorescence polymerization assay, we have discovered that the antibodies specific to non‐phosphorylated tau441 reduced tubulin polymerization. In the presence of non‐phosphorylated tau441, we observed successful tubulin polymerization even in the presence of anti‐tau antibodies. These studies indicate that tau plays a regulatory/competitive role in MT formationSupport or Funding InformationNIH/NIGMS/R15GM11905301