bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and Purification of Active S. commune Metacaspase Proteins
Authors: Jones Brandon M , Fox Kristin M 
Published: November 2023

Our objective is to produce purified active metacaspase proteins native to the fungus Schizophyllum commune using an E. coli expression system, compare their structural and functional properties, and understand the role of the N‐terminal prodomain in metacaspase function. Metacaspases are caspase homologs that participate in the clearance of protein aggregates, ER stress, and programmed cell death. Five S. commune genes encoding metacaspase proteins have been identified, Scp1–5. With the exception of Scp3 which lacks a prodomain, all other identified S. commune metacaspases contain an N‐terminal prodomain. While several of the metacaspase proteins have been expressed in E. coli, currently only one of these proteins, Scp3, displays activity upon overexpression. Production of active protein was accomplished by fusing Scp3 to the protein small ubiquitin modifying protein (SUMO), which has been shown to increase solubility of foreign proteins in E. coli.Currently, through PCR amplification and Gibson assembly, plasmid vectors encoding SUMO‐Scp1 and SUMO‐Scp5 fusion proteins without their respective prodomains (SUMO‐Scp1nopro and SUMO‐Scp5nopro) as well as SUMO‐Scp5 with its prodomain (SUMO‐Scp5pro) have been produced. E. coli have been transformed using these plasmids and after growth at 27°C, SUMO‐Scp1nopro, SUMO‐Scp5nopro, and SUMO‐Scp5pro have been produced based on SDS‐PAGE analysis. SUMO‐Scp1nopro has been partially purified using a nickel affinity column. This is the first time an S. commune metacaspase has been produced with an intact prodomain in soluble form.
Assessing Substrate Engagement by Single Molecules of the AAA+ Protease ClpXP
Authors: Saunders Reuben A. , Stinson Benjamin M. , Sauer Robert T. 
Published: November 2023

ClpXP, a AAA+ protease, binds ssrA‐tagged protein substrates, unfolds them, and translocates the polypeptide into a sequestered peptidase chamber for proteolysis. Although the sites of ssrA interaction in the ClpXP pore have been characterized, the dynamic steps by which initial binding leads to unfolding and degradation are poorly understood. Specifically, it is unclear whether initial binding is reversible or commits a substrate to unfolding and degradation. The kinetics of substrate degradation measured directly in population studies are also substantially slower than the kinetics expected from the single‐molecule rates of unfolding and translocation, raising the possibility that degradation initiation is a slow step in the ClpXP activity cycle.To clarify the kinetic mechanism of substrate engagement, we established a single‐molecule assay based on fluorescence quenching to assess substrate binding and release by ClpXP directly. A set of substrate proteins with a wide range of unfolding stabilities was labeled with a fluorescence quencher. We then bound fluorescently labeled ClpXP to a glass surface and used total internal reflection fluorescence microscopy to monitor single‐molecule fluorescence in the presence of different concentrations of substrate. The single‐molecule assay was validated by comparison with solution‐binding experiments.We observed many instances in which ClpXP binds and releases ssrA‐tagged substrates without degrading them. Commitment is a comparatively rare event and is slow or rate‐limiting in the kinetic process of degradation. Release without unfolding occurs more frequently for stably folded substrates. However, we also observe lengthy bound dwell times for stably folded substrates, which we identify as extended unfolding attempSupport or Funding InformationThis research was supported by the US National institutes of Health grant GM‐101988 (R.T.S.). R.A.S. was supported by the MIT Undergraduate Research Opportunities Program.
Mutational Editing of Strained New Protein Folds with Deletion Mutations
Authors: Pattengale Sarah R. , Kumirov Vlad K. , Szyszka Taylor N. , Cordes Matthew H.J. 
Published: November 2023

There are 1,200 known protein folds, some of which evolved from other folds. The consequences and causes of evolutionary fold switching are not well known. The fold switch that occurs in the Cro protein family is an example of new protein fold evolution. There are two major fold groups of Cro proteins: Cro proteins that have an ancestral all‐a fold and those that have a descendant a+b fold. Early forms of the a+b Cro fold, represented by prophage Pfl6 Cro protein, exhibited conformational strain in the turn connecting the first two helices. Two groups of a+b Cro proteins that emerged later show deletions in this turn, either in G14 or T15, with apparent relief of conformational strain. To determine whether a G14 deletion could account for the strain relief, we introduced a G14 deletion into the strained Pfl6 Cro using site‐directed mutagenesis. Using circular dichroism (CD), nuclear magnetic resonance (NMR), and fluorescence anisotropy (FA), we characterized the stability, structure, and DNA binding affinity, respectively, of this Pfl6 variant. The G14 deletion slightly increased the protein's thermal stability, partially relieved the strain in the helix 1–2 turn, and did not significantly change the DNA binding affinity compared to wild‐type Pfl6. These findings suggest that new protein folds may emerge through transitional forms that are edited through deletion and/or insertion mutations to remove awkward features.Support or Funding InformationThis research is funded by an NIH grant (R01 GM066806).
Structural analysis of CCT‐bound folding Intermediates for mTORC1 subunits Raptor and mLST8
Authors: Ludlam William Grant , Aoba Takuma , Cuéllar Jorge , Makaju Aman , Franklin Sarah , Valpuesta José M , Willardson Barry M 
Published: November 2023

The mTOR signalling complex 1 (mTORC 1) is a master regulator of cell growth and a high‐value drug target. While mTORC1 function has been studied extensively, little is known about how its subunits are folded and assembled together. Using a combination of cryo‐electron microscopy (cryo‐EM) and chemical crosslinking coupled with tandem mass‐spectrometry (XL‐MS), we investigated the mechanism by which the chaperonin CCT and its co‐chaperone, PhLP1, participate in the folding of mTORC1 subunits Raptor and mLST8. 2D cryo‐EM reconstructions of the raptor‐CCT complex show a mass completely filling the CCT folding cavity and extending out from it. This evidence supports the claim that CCT folds large proteins, such as Raptor, that do not fit inside its cavity by accommodating individual domains inside the cavity while the rest of the protein remains outside the cavity. 2D cryo‐EM reconstructions of the PhLP1‐mLST8‐CCT complex show that mLST8 sits inside the cavity while PhLP1 spans the cavity. XL‐MS data for the mLST8‐CCT complex indicate that mLST8 is oriented on one side of the cavity in contact the CCTα and CCTγ subunits. These results suggest different mechanisms for mLST8 and Raptor folding by CCT, one involving PhLP1 and the other involving a single domain Raptor.Support or Funding InformationGrants from the National Institute of Health: EY012287, GM07855
mRNA Quality Control Is Activated Due To Chromium (VI) Mediated Oxidative Damage
Authors: Garcia‐Doherty Naiara , Larson Abbi L , Six Marshall , Segal Scott P 
Published: November 2023

Chromium VI (Cr(VI)) is a genotoxic stressor that is known to cause 8‐oxoG bases to form in DNA. However, little is known regarding whether Cr(VI) has an effect directly at the level of mRNA. Prior data showed that treatment with Cr(VI) results in the selective assembly of P‐bodies, but not stress granules. This selective P‐body assembly requires translational repression to occur. This leads to a model in which mRNA either transcribed from damaged genes, or mRNA damaged directly by the Cr(VI) is sent to P‐bodies for degradation. To test between these two possibilities, strains in which transcription has been repressed by actinomycin D were subsequently treated with Cr(VI). Upon the dual treatment, P‐body assembly was unaffected, suggesting that Cr(VI) may have the ability to damage mRNA, and it is these mRNA that are, in turn, be localized to P‐bodies.Several mRNA quality control pathways exist in eukaryotes that potentially shuttle mRNA through P‐bodies for subsequent degradation. Of significance, are the Nonsense Mediated Decay (NMD) pathway, which degrades mRNA with premature termination codons, and the No‐Go pathway, which degrades mRNA in which ribosomes have stalled during translation. To test which pathway(s) may be activated in response to Cr(VI), deletion mutants were used to reduce or eliminate the activity of specific pathways. Upon reduction of NMD activity, no effect on P‐body assembly was seen in response to Cr(VI). However, reducing No‐Go decay, resulted in a significant reduction in P‐body assembly. Based on this data, it is likely that the increase in P‐body assembly through Cr(VI) can directly damage mRNA. This could potentially lead to stalled ribosomes during translation and subsequent activation of the No‐Go decay pathway.
Aim32p is a Novel Member of the Erv1 Translocation Pathway within the Mitochondrial Intermembrane Space of Saccharomyces cerevisiae
Authors: Dabir Deepa Vinay , Nguyen Kevin , Koehler Carla 
Published: November 2023

The mitochondrion is a key player in basic cellular homeostasis in eukaryotic cells, contributing to the synthesis of ATP, prevention of oxidative damage from reactive oxygen species and regulation of cell death. In yeast, most mitochondrial proteins are synthesized in the cytoplasm and imported post‐translationally into the organelle. One particularly interesting translocation pathway is the Erv1 oxidative folding pathway, a disulfide relay system comprising of Erv1 and Mia40 that couples the folding, oxidation and import of proteins into the mitochondrial intermembrane space (IMS). Besides import of proteins, Erv1 has diverse functions in the IMS; these include reoxidation of Mia40 upon release of the client protein, maturation of cytosolic iron‐sulfur cluster‐containing proteins and in the biogenesis of the TIM22 translocation pathway. Considering that Erv1 has several key functions in the IMS, we hypothesized that additional Erv1 partner proteins exist that function as electron acceptors and/or substrates. In this study, we elucidate the biological functions of Aim32p; a newly identified Erv1‐binding protein. Aim32p is an IMS‐ resident and under native conditions co‐exists in a complex with Erv1 and Osm1. Osm1 is a fumarate reducatase and has been recently postulated to function as an anaerobic electron acceptor of Erv1. Our structure predictions based on sequence analysis and homology modeling suggest that Aim32p is a putative thioredoxin‐ like ferredoxin protein. Although the function of most thioredoxin‐like ferredoxins remains unknown, the presence of a putative thioredoxin fold and a ferredoxin domain in Aim32p is highly suggestive of a role in redox sensing and electron transfer. Interestingly, loss of Aim32p also confers severe sensitivity to DNA damaging agents, MMS (Methyl methanesulfonate) and Hydroxyurea (HU). Thus, Aim32p may have a dual role in the mitochondrial IMS, participating in electron transfer reactions with Erv1 singularly or as a redox couple along with Osm1, and function in DNA damage stress response. Importantly, this study connects the redox‐regulated Erv1 translocation pathway to DNA damage stress response, and consequently its importance and versatility in several fundamental processes of the mitochondria.Support or Funding InformationThis work is supported by the Kadner Pitts grant.
Analysis of tectonin I and II and tectonin‐related proteins from Physarum polycephalum
Authors: Matsuda Takuya , Morita Rihito , Minami Yoshiko 
Published: November 2023

Physarum polycephalum, a true slime mold, is a lower eukaryote with a unique life cycle that consists of plasmodium, spore, amoeba, and sclerotium stages. The plasmodia consist of a huge, multinuclear cell that grows without cytokinesis. When the plasmodia are cultured in liquid medium, the cells segregate into numerous small‐walled masses called microplasmodia. The current study focused on the role of tectonin, a lectin‐like protein of the microplasmodia. Microplasmodia express tectonin I and II isoforms. Tectonin II has a larger molecular weight than tectonin I, and the two‐third of amino acid sequence of tectonin II is homologous to the entire sequence of tectonin I. Both tectonin I and II sequences contain six WD repeat motifs that form a beta‐propeller structure, a motif that is generally found in diverse proteins related to signaling processes. Tectonin‐like domains have been found in various types of proteins in a wide range of organisms. Examples include human tectonin (TECPR) and horseshoe crab galactose‐binding protein (GBP). The function of these tectonin‐like proteins has been well studied, whereas that of Physarum tectonin is not well characterized.We have previously reported that tectonin I is expressed during every stage of the Physarum cell cycle. However, haploid‐stage amoebas and spores do not express tectonin II. Therefore, it is believed that tectonin I and II may have distinct functions. Immunofluorescent intracellular localization has shown that tectonin I and II localize to plasmodia cell surface protrusions, suggesting that both proteins may function at the cell membrane and facilitate signal transduction, endocytosis, exocytosis, or other processes. To elucidate the function of tectonin, microplasmodia cell fractionation was performed. Although tectonin I and II were thought to be soluble proteins according to amino acid sequence analysis, both proteins separated into the membrane fraction. Solubilization experiments were performed using detergents, salts, ions, and various buffers to test protein release from the membrane surface. Tectonin I and II were released from the membrane when plasmodia were exposed to an alkaline buffer or to a buffer containing deoxycholate. Next, pull‐down assays were performed to analyze the presence of tectonin‐associated proteins using GST‐tectonin I and II followed by immunoprecipitation using anti‐tectonin I and II antibodies, respectively. Only a few proteins that putatively interacted with tectonin I and II were found. Finally, we confirmed that microplasmodia secrete tectonin‐like proteins in the culture medium during cultivation. We will present preliminary analyses of how tectonin interacts with tectonin‐related proteins.
CACN‐1 and its Interacting Proteins are Important Regulators of Somatic Development in Caenorhabditis elegans
Authors: Hugunin Julie 
Published: November 2023

With its easily visible U‐shaped gonad arm, Caenorhabditis elegans provides an excellent model to study germline development. This hermaphroditic nematode species has developed a mechanism to regulate the differentiation of its germline stem cells throughout development. C. elegans produce sperm until the onset of adulthood, when the worm permanently switches to producing oocytes. The Cram lab has identified the highly conserved protein CACN‐1 as an important player in proper germline differentiation. Loss of CACN‐1 leads to a masculinization of the germline where gonad arms are either exclusively filled with sperm, or contain a few irregular oocytes in addition to sperm. Previously, the lab identified CACN‐1 interacting proteins using tandem affinity purification and proteomics (TAP)1. CACN‐1 interactors grouped into a novel network suggest that CACN‐1 interacts with the spliceosome1. Depletion of some of these interactors in wild type animals also caused germline defects in differentiation and development comparable to CACN‐1. DAPI, a nuclear stain, revealed that gonad arms that lacked CACN‐1 and many of its interactors exhibited endomitotically duplicating oocytes (Emo phenotype) in a significant number of gonad arms containing abnormal oocytes. Due to the link between the Emo phenotype and ablated sheath cells2 an actin stain was used to examine sheath cell presence and germline phenotypes when these proteins were depleted via RNAi. As expected depletion of CACN‐1 and its interacting proteins lead to defects in sheath actin staining that correlated with Emo and sperm containing gonad arms. In addition, loss of these proteins in a worm strain that is only susceptible to RNAi in the somatic cells caused germline defects comparable to those observed in wildtype animals. Our data suggests that this novel network of proteins plays an essential role in somatic gonad development that has a direct effect on germline differentiation and maintenance. Ultimately a better understanding of CACN‐1 and its network of proteins in C.elegans may elucidate the role of splicing and translational regulation of gene expression in other processes, including disease and development, across species.Support or Funding InformationA special thanks is extended to Alyssa Cecchetelli, Michael Doherty and Erin Cram, as well as the entire Cram Lab, Northeastern University's Biology Department for supporting this research, and the NIH (R01 GM85077) for funding.
Transcriptional complexes involving PAX3‐FOXO1
Authors: Weatherspoon Amber , Cherlin Tess , Johanson Kelly 
Published: November 2023

Rhabdomyosarcoma is a rare soft tissue cancer that most often affects children and young adults but can occur at any age. Of the three types of Rhabdomyosarcoma, Embryonal, Alveolar, and Anaplastic, we focus on Alveolar Rhabdomyosarcoma (ARMS). Patients with ARMS have a chromosomal translocation that results in the production of a fusion protein, PAX3‐FOXO1. Although there are other translocations present in ARMS patients, PAX3‐FOXO1 is the most common and is liked with a more aggressive disease phenotype. 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 close proximity. With the knowledge that protein‐protein interactions involving FOXO1 have been shown to contribute to DNA targeting, we then examined the regions surrounding these dual motifs for the presence of common transcription factor binding sites. A number of common recognition sites, including those for MYOD, AP4R, and NEUR were overrepresented as compared to the expected distribution of these sequences in the genome. We are currently examining the contribution of these and other transcription factors to PAX3‐FOXO1 DNA‐binding through several in vitro assays. Identification of PAX3‐FOXO1 binding partners will give more detail about the mechanism of PAX3‐FOXO1 targeting and gene activation.Support or Funding InformationNIH SC2GM095430UNCF Stem Scholars Program (USSP)NIH‐BUILD (TL4MD009637)
Competition Between Tamm‐Horsfall Protein and Heparin in Ligand Interactions
Authors: Haskell Matthew , Rhodes Diana C.J. 
Published: November 2023

Tamm‐Horsfall protein (THP), also called uromodulin, is an 86kD acidic, single‐chain, glycoprotein produced in the kidney and excreted into the urine. THP binds various molecules of the immune system including immunoglobulins (Ig), complement 1q (C1q), and factor H (fH). Heparin is a negatively‐charged, sulfated polysaccharide that binds to a wide range of proteins, including C1q, fH, antithrombin III (ATIII), basic fibroblast growth factor (FGF2), and interleukin‐10 (IL‐10). The purpose of this study was to determine if heparin competed with THP for binding to proteins that interact with both THP and heparin (i.e. C1q and fH), and to screen other heparin‐binding proteins for previously unidentified interactions with THP. Dot blots were performed by immobilizing 1 μg of C1q, fH, and bovine serum albumin (BSA) to 4 strips of nitrocellulose (NC). One NC strip was stained with amido black and the other 3 NC strips were incubated with either THP (10 μg/ml), THP (10 μg/ml)/heparin (20 μg/ml) (sodium salt from porcine intestine), or no THP/heparin, before incubation with sheep anti‐THP antiserum and then donkey anti‐sheep IgG ‐ HRP. These blots showed that THP bound strongly to C1q and fH, and that this binding was inhibited by heparin. Heparin also inhibited the background binding of the antisera to C1q. ELISAs were used to quantify the ability of heparin to inhibit C1q (5 μg/ml) binding to immobilized THP (40 μg/ml). A steep inhibition curve was obtained, with no inhibition of C1q binding to immobilized THP occurring with 0.02 units/ml heparin, 50% inhibition of binding occurring with between 0.04 and 0.05 units heparin/ml, and 90% inhibition occurring with 0.1 unit heparin/ml (170 units/mg heparin). Control wells showed that heparin's inhibition of C1q binding to THP was not due to inhibition by heparin of the antisera's binding to C1q. Additionally, dot blots were used to evaluate binding of soluble THP to ATIII, IL‐10, and FGF2, three of the many proteins that heparin has been shown to bind. These blots demonstrated that THP bound to FGF2 and IL‐10, and this interaction was blocked by the addition of heparin. THP did not bind ATIII. In summary, heparin inhibited THP's binding to NC‐immobilized C1q and fH, as well as inhibiting immobilized THP's binding to soluble C1q in ELISAs. Protein‐protein interactions between THP and FGF2 and IL‐10, that were sensitive to heparin inhibition, were discovered. Heparin and THP coexist in the urine, especially after intravenous heparin administration or when heparin is instilled into the bladder as a therapy for interstitial nephritis. Mast cells are prominent in the wall of the urinary bladder in interstitial cystitis, so competition of heparin and THP for binding to other proteins may be important in this disease. Further studies to assess the physiological significance in THP/heparin competition for binding to various proteins is warranted.Support or Funding InformationFunded by the PNWU Research Seed Program.
Investigating the Activation of the Tm2‐2 Immune Receptor in Response to the Tobamovirus Movement Protein
Authors: Sandoval Carina , Sacco Melanie A. 
Published: November 2023

Effector‐triggered immunity (ETI) is a plant defense mechanism that evolved to detect pathogen effector proteins and initiate immune responses. Effectors are recognized by a direct or indirect interaction with plant immune receptor proteins. ETI often limits pathogen proliferation by promoting hypersensitive response (HR), a type of programmed cell death. In tomato (Solanum lycopersicum), the Tm2‐2 immune receptor provides resistance to Tobamovirus 30kDa movement protein. Tm2‐2 belongs to the CC‐NBS‐LRR class of resistance proteins, for which activation is mediated by specific intramolecular domain interactions. To study Tm2‐2 activation, mutations were introduced into the full‐length and truncated CC (coiled‐coil) domain clones. Highly conserved threonine or serine residues at positions 7, 8 and 13 were identified as potential phosphorylation targets and were mutated to aspartic acid (T7D, S8D, S13D) or to alanine (T7A, S8A, S13A). Nicotiana benthamiana was used for transient expression of constructs to observe for the effects of mutations on HR. Expression of the truncated CC domain clones with T7A or T7D mutations did not induce the HR in the presence or absence of the viral effector protein. The CC domain of the immune receptor with the effector protein is not sufficient to activate the plant's immune response. The full‐length T7D clone failed to elicit HR in the presence of the effector protein, showing loss of function. Unexpectedly, the full‐length T7A clone retained function and triggered HR in the presence of the effector protein. This evidence supports our hypothesis that position 7 of Tm2‐2 is a phosphorylation target that negatively regulates the activation of the Tm2‐2 immune receptor such that Tm2‐2 is inhibited in the charged state, but when neutral, Tm2‐2 is competent for activation of plant defenses. Understanding the mechanism of activation for Tm2‐2 can inform future engineering of efficient immune receptors that confer resistance against various pathogens.Support or Funding InformationThis project is supported by the Howard Hughes Medical Institute undergraduate research grant.