bluuebunny/crossref_metadata_2025_split huggingface dataset:
Repeat expanded Ataxin‐1 mRNA and protein is co‐regulated at PML bodies
Authors: Fanslow Danielle , Cogswell Alexis , Strojny Chelsee , Garza‐Gongora Arturo , Smith Erica , Kosak Steven 
Published: November 2023

The trinucleotide repeat disorder Spinocerebellar Ataxia‐1 (SCA‐1) is a neurodegenerative disease characterized by progressive motor neuron deterioration. The disease is caused by an expansion of the CAG tract in the gene ATXN1, resulting in highly structured ATXN1 mRNAs and misfolded Ataxin‐1 protein aggregates. It is becoming clear that aggregation‐prone expanded mutant Ataxin‐1 proteins are degraded through a nuclear protein homeostasis mechanism. However, the regulation of expanded mutant ATXN1 mRNA remains unclear. In the current study we use a mutant Ataxin‐1 with an expanded polyglutamine tract (85Q‐Atxn1) as a model of altered mRNA structure and proteotoxic stress. Our group hypothesizes that nuclear polypeptide synthesis is integrated with a cellular response to stress, specifically responding to the presence of aberrant mRNA secondary structures in the nucleus. Interestingly, we detect nascent polypeptides labeled by puromycin (puro) associated with 85Q‐Atxn1 inclusion bodies as well as ATXN1 mRNA. 85Q‐Atxn1 with an associated puro signal significantly localizes to promyelocytic leukemia (PML) nuclear bodies. PML bodies are multifunctional subnuclear organelles consisting of PML protein and a heterogeneous composition of protein binding partners as well as nucleic acids. PML body composition dynamically changes in response to Atxn1 expression, as shown by a proximity labeling proteomics analysis. Altered PML expression changes puro signal intensity as well as 85Q‐Atxn1 accumulation, suggesting a protective role of PML during expression of 85Q‐Atxn1. Additionally, ribosomal stalk proteins significantly associate with PML and 85Q‐Atxn1. The No‐go decay (NGD) mRNA surveillance mechanism is necessary for the protection against the accumulation of 85Q‐Atxn1 and correlates with PML localized puro accumulation. Overall, our analysis has uncovered a novel regulatory phenomenon in which aberrant mRNAs, protein synthesis, and protein degradation are functionally integrated at PML bodies.Support or Funding InformationFunding by CMBD Training Grant‐NIH T32 GM08061, NIH DP2 OD008717 to S.K, NIH F32CA189413 to A.A.C, and CBC PDR‐003 to S.K and A.A.C.
Molecular mechanism of JNK attenuation by parkin
Authors: Davis Cameron Wayne , Zhan Xuanzhi 
Published: November 2023

Parkinson's disease (PD) is characterized by the disappearing pigmentation, accumulation of lewy bodies, and the ultimate degeneration of neurons in the substantia nigra pars compacta region of the brain. The most prevalent indication of hereditary PD is a mutation in the PARKIN gene. Parkin facilitates the proteasomal degradation of target proteins through its ubiquitination‐catalyzing activity. The E3 ubiquitin ligase has been further implicated in attenuating the activation of the c‐jun N‐terminal kinase (JNK) pathway, a pro‐apoptotic mitogen‐activated protein kinase (MAPK) signaling cascade, implying neuroprotective characteristics. However, the mechanism whereby parkin regulates the JNK pathway remains obscure. Utilizing in vitro pull‐down assays in tandem with co‐immunoprecipitation methods, we have identified a novel interaction between parkin and apoptosis signal‐regulating kinase (ASK‐1), an upstream kinase in the JNK pathway. This interaction was characterized through co‐overexpression in HEK293T cells, which indicated the parkin‐induced degradation of ASK1. In vitro ubiquitination assays showed an enhanced activation of parkin in the presence of ASK1. Our results suggest the interaction with ASK1 is able to disrupt the intrinsic auto‐inhibition of parkin, enhancing its activation to carry out the ubiquitin‐signaled degradation of ASK1.Support or Funding InformationTennessee Technological University Chemistry Department Student Research Grant
Proteasome Activation via a Functional Switch of the Rpt6 C‐terminal Tail Following Chaperone‐dependent Assembly
Authors: Park Soyeon , Li Frances , Sokolova Vladyslava 
Published: November 2023

In the proteasome, the proteolytic 20S core particle (CP) associates with the 19S regulatory particle (RP) to degrade polyubiquitinated proteins. Six ATPases (Rpt1‐Rpt6) of the RP form a hexameric Rpt ring and interact with the heptameric α ring (α1–α7) of the CP via the Rpt C‐terminal tails individually binding to the α subunits. Importantly, the Rpt6 tail has been suggested to be crucial for RP assembly. Here, we show that the interaction of the CP and Rpt6 tail promotes a CP‐Rpt3 tail interaction, and that they jointly mediate proteasome activation via opening the CP gate for substrate entry. The Rpt6 tail forms a novel relationship with the Nas6 chaperone, which binds to Rpt3 and regulates the CP‐Rpt3 tail interaction, critically influencing cell growth and turnover of polyubiquitinated proteins. CP‐Rpt6 tail binding promotes the release of Nas6 from the proteasome. Based on disulfide crosslinking that detects cognate α3‐Rpt6 tail and α2‐Rpt3 tail interactions in the proteasome, decreased α3‐Rpt6 tail interaction facilitates robust α2‐Rpt3 tail interaction that is also strongly ATP‐dependent. Together, our data support the reported role of Rpt6 during proteasome assembly, and suggest that its function switches from anchoring for RP assembly into promoting Rpt3‐dependent activation of the mature proteasome.Support or Funding InformationBoettcher Foundation
Nitrogen Starvation and Rapamycin both Induce Autophagic Degradation of Proteasome Complexes
Authors: Roelofs Jeroen , Waite Kenrick A , Vontz Gabrielle , De La Mota‐Peynado Alina 
Published: November 2023

Under conditions of nitrogen starvation yeast cells need to degrade proteins to obtain nutrients that can be dedicated to the synthesis of proteins involved in crucial cellular processes. Two systems in the cell are responsible for the degradation of most cellular proteins, the ubiquitin‐proteasome system and the lysosome/vacuole, and thus could contribute to this process. The proteasome is a 2.5 MDa complex that consists of a core particle (CP) that harbors the proteolytic active sites within a cylindrical structure, and one or two regulatory particles (RPs), that control substrate binding, unfolding, and entry of substrates into the CP. We reported recently that proteasomes themselves are degraded upon prolonged nitrogen starvation. Using GFP‐tagged proteasome subunits we showed proteasomes are targeted to the vacuole and that this depends on the core autophagy machinery. This degradation, however, does not simply reflect general bulk autophagy of cytosolic proteasomes, because we observed that nuclear proteasomes are exported out of the nucleus upon nitrogen starvation. Furthermore, starvation induces a dissociation of proteasomes into CP and RP. Both subcomplexes are targeted to the vacuole, but depend on different factors; only CP autophagy required the deubiquitinating enzyme Ubp3. Ubp3 is not required for bulk autophagy, further confirming the model that proteasomes are specifically targeted to the vacuole. The next goal of our lab is to understand the signaling pathways and mechanisms that are responsible for the nuclear export and vacuolar targeting of proteasomes. One major signaling pathway inhibited upon nitrogen starvation is the target of rapamycin complex 1 (TORC1) pathway. Surprisingly, it was recently reported by Rousseau and Bertolotti (Nature, 2016) that an inhibitor of this pathway, rapamycin, actually induces an upregulation of proteasomes. Similar to their data, we observe an initial upregulation of proteasome levels and activity upon rapamycin treatment. However, prolonged exposure results in vacuolar targeting and degradation of proteasomes similar to what we reported upon nitrogen starvation. We observed several differences between nitrogen starvation and rapamycin treatment, indicating the autophagic degradation of proteasomes upon nitrogen starvation does not solely depend on the mTORC1 pathway.Our work demonstrates that in addition to the well‐characterized transcriptional upregulation of genes encoding proteasome subunits, cells are also capable of down regulating cellular levels of proteasomes by targeting them for autophagy. Characterizing the pathways responsible for degradation of proteasomes upon nitrogen starvation or under conditions of superfluous, damaged, or misassembled proteasomes will be important to understand how cells regulate proteolytic capacity and protein homeostasis in the cell.Support or Funding InformationNIH‐NIGMS 1R15GM112142 and R01GM118660
Identifying a serine protease network involved in ovarian cancer progression using activity‐based protein profiling (ABPP)
Authors: Mehner Christine , Hockla Alexandra , Radisky Derek C , Radisky Evette S 
Published: November 2023

Ovarian cancer remains a devastating disease with 5 year survival rate in advanced stage of only 27%. Advanced stage tumors metastasize through continuous shedding of tumor cells into the peritoneal fluid, aided by the resistance of these cells to anoikis, a form of apoptosis normally triggered by loss of cell‐matrix interactions. We have previously shown that the serine protease inhibitor SPINK1 activates anoikis resistance in ovarian cancer cells via its protease inhibitory function; however, the specific relevant protease target(s) remain unknown. To find these targets, we have synthesized an activity‐based protein probe with a diphenylphosphonate warhead that covalently modifies serine proteases, with greatest specificity towards trypsin‐like proteases. We have used this probe to identify ovarian cancer cell secreted and membrane‐bound serine proteases possessing differential activity in the presence versus absence of SPINK1, using a proteomics approach. We are evaluating candidate proteases for involvement in anoikis resistance in ovarian cancer models. Our studies implicate a serine protease network in regulation of anoikis in ovarian cancer cells. By identifying critical proteases that control anchorage‐independent viability in ovarian cancer metastasis, we aim to elucidate important downstream pathways and ultimately develop therapeutic approaches that can intervene in mechanisms of anoikis resistance.Support or Funding InformationMayo Clinic Graduate School, Rochester MN
Structural and Biochemical Insights into the Activation and Substrate Selectivity of Clostripain‐like Proteases Secreted From Commensal Gut Bacteria
Authors: Roncase Emily J , O'Donoghue Anthony J , Wolan Dennis W 
Published: November 2023

Commensal gut bacteria secrete a broad variety of biological and chemical signals to influence intestinal homeostasis and modulate immune responses, including active proteases. Enteric cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria and they play a crucial role in intestinal inflammation as they cleave host proteins, activate cell surface receptors, and disrupt epithelial barrier integrity. Recently, members of the clostripain‐like (C11) family of cysteine proteases were shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. We have structurally and functionally characterized two additional members of this family from the predominant gut symbiont Bacteroides thetaiotaomicron: BT1308 and BT0727. These secreted proteases are predicted to be packaged into outer membrane vesicles capable of interacting with host epithelial cells and are known to be activated through internal cleavage events. We verified the necessity of self‐cleavage for maturation, showed this cleavage event occurs in trans, and identified the sites of cleavage following designated arginine residues. Using Multiplex Substrate Profiling by Mass Spectrometry we identified a unique cleavage motif of X‐X‐T/S‐K/R common among C11 proteases and determined protease‐specific differences in P3–P4 recognition. To supplement this biochemical data, we solved the structures of both active enzymes in complex with a tetrapeptide inhibitor at under 2.4Å resolution, and these structures highlighted subtle variations in active site architecture that confer selectivity in substrate recognition. Furthermore, comparing our structures to an inactive mutant of a C11 protease from Parabacteroides distasonis revealed that proteolytic activity could be dependent on a conformational change that reorients the active site histidine upon self‐cleavage. Our findings provide insight into the mechanism and specificity of C11 protease activity and will aid in the rational design of protease‐specific probes that can be used to interrogate the role of C11 proteases in inflammation and bacterial fitness.Support or Funding InformationBoehringer Ingelheim, National Science Foundation
The effect of disrupting a distal hydrogen bond network on serine protease function
Authors: Villa Rodolfo , Baird Teaster 
Published: November 2023

Engineered serine proteases constitute a distinct therapeutic class with a potential for diverse clinical applications. One main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. However, if a protease is to be effective as a therapeutic agent, it needs to display resistance towards inhibition. Given that the majority of protease therapeutics currently used are of the trypsin fold, trypsin itself serves as an ideal structure‐function relationship model to address this issue. Previous studies, using small synthetic peptides, bovine pancreatic trypsin inhibitor (BPTI) and an ecotin variant (M84R ecotin) with engineered trypsin variants have shown that prime side interactions can affect association with macromolecular inhibitors through interaction with residues at positions 39 and 60. The K60A trypsin variant (K60A‐Tn) had the lowest amount of free enzyme in equilibrium inhibition studies while using BPTI, but the opposite was observed with M84R ecotin. There is evidence that increased mobility of Y39 caused the K60 substitution to impact P4′ interactions and P2′ hydrogen bond, resulting in altered trypsin:inhibitor interactions. Specifically, replacing lysine‐60 with alanine does not allow the K60 and Y39 charged hydrogen bond interaction regardless of the P4′ hydrogen bond presence, suggesting that a polymorphism at position 60 may influence interaction between Y39 and inhibitors. However, given that BPTI and ecotin are also proteins, their interaction with trypsin likely reflects the interaction that serine proteases will have with natural protein substrates. Therefore, a desired weakened interaction between the engineered trypsin and the inhibitor may also suggest a similar weakened interaction between the engineered trypsin and protein substrates which cannot be observed with the small peptide substrates that were used previously. Our focus in this study is to use a protein as a substrate, anticipating insight on the enzyme‐substrate interaction via catalytic activity of K60A‐Tn compared to wild‐type trypsin. Without the K60 and Y39 charged hydrogen bond interaction, we predict to see weakened substrate affinities, causing lowered catalytic efficiency.Support or Funding InformationDr. Teaster Baird National Institute of Health (NIH)
Site‐Directed Mutagenesis of Recombinant Aedes aegypti Trypsin‐like Serine Proteases II and IV (AaSPII and AaSPIV)
Authors: Eilerts Diane F , Rascon Alberto A 
Published: November 2023

Aedes aegypti mosquitoes act as vectors of Dengue fever, Zika, yellow fever, and Chikungunya viruses. The female Ae. aegypti mosquito uses blood meal proteins to obtain nutrients required for the egg development process. Inhibition of specific proteases responsible for blood meal digestion may allow for novel methods of vector control. Characterization of newly identified Ae. aegypti serine proteases such as AaSPII and AaSPIV is necessary to gain insight into the complex mechanism of mosquito blood meal digestion. Recombinant expression of Ae. aegypti trypsin‐like serine proteases II and IV (AaSPII and AaSPIV) was achieved in preparation for protein purification. Preliminary results obtained from growth experiments indicate that these proteases are likely autocatalyzing and autodigesting. Site‐directed mutagenesis was successfully performed in order to introduce a point mutation at the active site serine residue to an alanine. This change should yield an inactive protease and help determine if our hypothesis of autocatalytic activation is true. Future studies will focus on purification of inactive recombinant enzyme and structural studies.Support or Funding InformationSC3 Research Grant from NIH
Overexpression and Characterization of the rquA Gene Product Involved in the Biosynthesis of Rhodoquinone
Authors: Zander Alison , Shepherd Jennifer 
Published: November 2023

Infections caused by parasitic helminths are sometimes referred to as “the great neglected tropical diseases.” Endemic to many countries, including the United States, parasitic helminths are at the origin of the diseases most commonly diagnosed in developing countries. Affecting both humans and livestock, these helminths are the target of extensive research as their genome is further elucidated. The drug products that currently exist to combat infections are subject to parasitic resistance; therefore, the race to discover different drug‐targets specific to these parasites is essential. In an effort to develop an answer to this problem, we have been researching the rhodoquinone (RQ) biosynthetic pathway. RQ is the final electron transporter in the anaerobic fumarate reductase pathway, which occurs in the mitochondrial and plasma membranes. Although its biosynthetic pathway is still largely unknown, it has been discovered that the gene known as rquA is required for RQ biosynthesis in the model organism, Rhodospirillum rubrum. Expression and purification of the gene product of rquA (RquA) could provide a target for inhibition of RQ biosynthesis. Additionally, determining the crystalline structure and any proteins that are in a complex with RquA could further information in development of a novel drug. After isolation and purification of RquA from overexpression in E. coli, a pull‐down experiment was performed using nickel‐coated magnetic beads to bind the protein (containing a hexahistidine tag). Following this, the bead protein complex was combined with lysate collected from solubilized wild‐type R. rubrum membranes. Providing these conditions for the protein allowed RquA to complex with any other proteins that it may interact with in its normal state. Upon SDS‐PAGE gel electrophoresis, and staining with Coomassie blue, there was evidence of at least 4 proteins that appear to be associated with RquA.Support or Funding InformationNational Institutes of Health (1R15GM096398‐01) and Howard Hughes Medical Institute (award to Gonzaga University)
The Structure and Function of Sulfite Reductase Flavoprotein: Three Faces of a Dynamic Enzyme
Authors: Askenasy Isabel , Andrews Rachel , Stroupe M. Elizabeth 
Published: November 2023

In enterobacteria like Escherichia coli, NADPH‐dependent assimilatory Sulfite Reductase (SiR) is the central enzyme in the sulfur assimilation pathway. SiR performs the six‐electron reduction of sulfite (SO3−2) to sulfide (S−2). SiR is a soluble multimeric enzyme with two subunits that combine in a 2:1 ratio to form a stable dodecameric complex. The α subunit (SiRFP) is a cytochrome P450 oxidoreductase (CYPOR) homolog. The β subunit (SiRHP) is a monomeric metalloenzyme with a Fe4S4 cluster and a porphyrinoid group called siroheme that forms the active site for the complex. SiRFP has the prototypical, three‐domain architecture of CYPOR that transfers electrons from the cellular donor NADPH to its FAD and FMN cofactors sequentially, ultimately delivering them to the binding partner, in this case SiRHP. However, SiRFP differs from other CYPORs in several ways: 1) SiRFP is a soluble protein that forms a homo‐octamer when expressed in the absence of SiRHP and 2) SiRFP forms a stable complex with SiRHP. The main aim of our research is to understand the electron transfer between the subunits in the context of the unique stoichiometry and structure of the complex. We identified two regions of SiRFP whose solvent accessibility changes upon assembly with SiRHP. The two regions of the structure are on different faces of the protein: one is in the N‐terminal FMN‐binding domain and the other is in the C‐terminal NADPH binding domain. Electrons are known to pass from the FMN binding domain to SiRHP. Interestingly, the interface that is part of the NADPH binding domain is sufficient to form a tight complex with SiRHP, suggesting the interface within the FMN binding domain is transient. To test this hypothesis, we synthesized three point variants: Y101A, Q191K, and V500D. Y101 is part of the FMN binding domain. Q191 plays a role in the conformational change that is known to govern electron transfer in CYPOR. V500 is part of the SiRFP region that interacts with SiRHP. Then, we used complementation assays and isothermal titration calorimetry (ITC) to probe the effect of these mutations. The three combined mutations has a synergic effect, resulting in cells that were unable to grow in minimal media. The ITC data showed that only the SiRFP V500D and variants combined with it had a reduced affinity for SiRHP respect to the wild‐type enzyme. From these results we conclude that SiR complex formation is driven by hydrophobic interactions in the C‐terminus of SiRFP, far from the site of electron transfer to its SiRHP partner. In contrast, transient interactions involving Y101 are important for electron transfer from the FMN cofactor to the metallic cofactors in SiRHP.Support or Funding InformationMCB1149763
Cofactor Analysis of Nicotinate Dehydrogenase and 6‐Hydroxynicotinate Dehydrogenase in Bacillus niacini
Authors: Sherer Laura Ashley , Snider Mark Ashley , Pandelia Maria‐Eirini , Rowlett Roger 
Published: November 2023

Prevalent in many pharmaceuticals, dyes, and herbicides, N‐heterocyclic aromatic compounds have recently been recognized as environmental contaminants of emerging concern. Understanding microbial degradation of N‐heteroaromatics may inform bioremediation efforts to remove or neutralize these contaminants. Nicotinic acid (NA, vitamin B3) is considered a model compound, and consequently, studying bacterial catabolism of NA may help elucidate the degradation of N‐heteroaromatics as a whole. In this study, we investigated nicotinate dehydrogenase (NDH) and 6‐hydroxynicotinate dehydrogenase (6‐HDH) from Bacillus niacini, an aerobic soil bacterium. These enzymes hydroxylate NA and 6‐hydroxynicotinic acid (6‐HNA), respectively, catalyzing the first two steps of NA catabolism. To accomplish this chemistry, computational predictions suggest that NDH and 6‐HDH each contain two [2Fe‐2S] clusters, a molydopterin cytosine dinucleotide (MCD), and a FAD coenzyme. The subunits of these enzymes were purified from E. coli and analyzed for their respective cofactors. The [2Fe‐2S] cluster subunit was found to contain 3.9 mol of iron, and the FAD subunit was visibly yellow and found to contain FAD as predicted. In the purified molybdenum subunits, preliminary investigations suggest a 1:10 occupancy of MCD. In order to increase this occupancy, ongoing studies are using Pseudomonas strains as the recombinant host.Support or Funding InformationAcknowledgments: Whitmore‐Williams Science Scholarship, the Clare Boothe Luce Program, and the Department of Chemistry at The College of Wooster