bluuebunny/crossref_metadata_2025_split huggingface dataset:
The degradation requirements for topologically distinct quality control substrates in the yeast endoplasmic reticulum
Authors: Guerriero Christopher James , Reutter Karl Richard , Augustine Andrew , Brodsky Jeffrey L. 
Published: November 2023

In eukaryotic cells, nearly one third of all newly synthesized proteins are translocated into the endoplasmic reticulum (ER). Inside the ER, proteins are post‐translationally modified and folded by molecular chaperones into their native states. Mutations, transcriptional/translational errors, aberrant post‐translational modifications, or misassembly can result in ER‐associated degradation (ERAD), a quality control pathway which recognizes and degrades misfolded proteins to prevent toxic gain‐of‐function phenotypes. Given the vast array of conformers that proteins adopt, it is not surprising that many diseases result from protein degradation by the ERAD pathway.The rate‐limiting step during ERAD is substrate retrotranslocation from the ER into the cytoplasm, which occurs prior to proteasome‐mediated degradation. Therefore, the retrotranslocation step can tune degradation efficiency and potentially be targeted to mitigate ERAD‐related diseases. Retrotranslocation is driven by the AAA‐ATPase Cdc48p in yeast (p97 in mammals), which extracts integral membrane proteins either directly from the ER membrane or through an as‐yet‐elusive retrotranslocation channel. Although integral membrane proteins have diverse membrane spanning domains and orientation, little is known about how the placement of a misfolded domain in the ER lumen versus the cytoplasm influences ERAD efficiency. We predict that extraction of a large ER lumenal domain will slow retrotranslocation kinetics.To test this hypothesis, a pair of model ERAD substrates were designed to contain the same truncated nucleotide‐binding domain from a well‐characterized ERAD substrate, Ste6p*, but on opposite sides of the ER membrane. We previously demonstrated that a soluble version of this domain, NBD2*, when expressed in the cytoplasm is degraded by the proteasome, and therefore can be used as a transposable degron (Guerriero et al., JBC, 2013). Our first model substrate, Chimera A*, is a dual pass membrane protein fused to NBD2* positioned in the cytoplasm. By contrast, Chimera N*, is a single pass membrane protein which deposits NBD2* in the ER lumen. Initial characterization confirms the proper topology of these substrates and proteasome‐dependent degradation. Moreover, the substrates rely on ER resident E3 ligases, Hrd1p and Doa10p, for efficient degradation. We are currently determining the relative dependence on Cdc48p for both degradation and retrotranslocation in order to better understand the energetic barriers to ERAD (Supported by NIH grants DK101584 to C.J.G. and GM075061 to J.L.B.).
A High‐Content RNAi Screen to Elucidate Novel Effectors of Cardiac Proteotoxicity
Authors: McLendon Patrick M , Davis Gregory , Gulick James , Shay‐Winkler Kritton , Molkentin Jeffery D , Robbins Jeffrey 
Published: November 2023

RationaleProteotoxicity has emerged as an important underlying pathologic mechanism in cardiac disease. Many heart failure phenotypes, including ischemia/reperfusion, pressure overload‐induced hypertrophy and desmin‐related cardiomyopathy accumulate misfolded protein within the cytoplasm of cardiomyocytes, potentially leading to cell death and dysfunction. While recent success in treating these phenotypes has been achieved by inducing the protein degradation mechanisms that mediate protein quality control in cardiomyocytes, more study is needed to understand the players and processes involved in pathologic aggregation.ObjectiveTo uncover, in a non‐biased way, novel gene targets that reduce protein accumulation in a cellular model of proteotoxic heart disease.Methods and ResultsTo address this, we developed a cellular model of cardiac protein aggregation in primary mouse cardiomyocytes by expressing a fluorescent aggregation‐prone protein, a mutated αλπηα−B‐crystallin (CryABR120G). The cells developed large intracellular aggregates, consistent with data from mice and humans. We subjected this cell model to a high‐content, genome‐wide RNAi knockdown screen using a lentiviral shRNA library (~ 16,000 genes). The effect of gene knockdown was calculated by measuring the quantity of punctate aggregate structures within cardiomyocytes. Using shRNA knockdown, we identified 236 genes in the mouse genome that could substantially ( > 50%) reduce aggregate content in cardiomyocytes. The hits span diverse and interesting cellular processes including mitochondrial and apoptotic cell death, calcium signaling, post‐translational modifications and metabolism and suggest overlapping therapeutic mechanisms between proteotoxicity and other cardiovascular pathologies.ConclusionsWhile rigorous validation of our candidate list is ongoing, we have confirmed several candidates which can lead to reduction of proteotoxic inclusions in cardiomyocytes. We are confident that further interrogation of our candidate genes will contribute vital details on the molecular mechanisms of pathologic protein aggregation, as well as yield new and interesting therapeutic targets.Support or Funding InformationThis work is supported by funding from the National Institutes of Health (to PMM and JR) and by Le Fondation Leducq (to JR)
Development of Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Escamilla Yaritza , Hughes Casey , Hu Yanmei , Bullard James M 
Published: November 2023

IntroductionPseudomonas aeruginosa is a common cause of nosocomial infections and the leading cause of mortality in patents with cystic fibrosis. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis.ResultsP. aeruginosa glutaminyl‐tRNA synthetase (GlnRS) was overexpressed in E. coli cells, and purified to homogeneity. GlnRS from P. aeruginosa is a discriminating synthetase with respect to the requirement for the presence of tRNAGln to produce a stable glutaminyl‐AMP intermediate. When compared with GlnRS from E. coli the amino acid sequence of GlnRS from P. aeruginosa are only 60% conserved. The kinetic parameters for interaction with tRNA were determined and the KM and Vmax were 1.0 μM and 0.23 μM min−1, respectively. The observed kcat was calculated to be 0.15 s−1 resulting in a kcat/KM value of 0.15 s−1μM−1. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa GlnRS in a high throughput format. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with the ability to inhibit function of the enzyme. Three compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM02E04, BM04B05, and BM04H03 inhibit the activity of P. aeruginosa ArgRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. All three of these compounds exhibited promising MICs against Gram+ bacteria and moderate activity against Gram‐ bacteria causing respiratory infections.ConclusionGlnRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.Support or Funding InformationResearch was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017) and from the NIH UTPA RISE program grant # 1R25GM100866‐01.
Knockdown of axonemal inner arm dynein protein IC138 Trypanosoma brucei causes flagellar detachment and defects in motility
Authors: Marchese Anthony , Wilson Corinne , Springer Amy L. 
Published: November 2023

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Motility is critical for its life cycle progression and pathogenesis. However, the regulation of flagellar function is not well understood. T. brucei utilizes a flagellum that attaches along the length of the cell, beating from tip‐to‐base to move the cell forward. Studies have found that the assembly and regulation of proteins within the axoneme, the microtubule‐based cytoskeleton of the flagellum, are well conserved. Within the axoneme are outer and inner arm dyneins, structures known to be involved in microtubule sliding which drives cell motility. Inner arm dynein I1/f has been found to play an important role in regulation of the flagellar beating. We are interested in the characterization of T. brucei IC138, an intermediate chain of dynein I1/f. This protein has been has been studied in green alga Chlamydomonas reinhardtii and shown to be a phosphoprotein important for the regulation of dynein I1/f. A T. brucei strain was created to inducibly target IC138 by RNA interference. By 72‐hours post‐induction, quantification of the knockdown revealed that IC138 protein levels were reduced by 87%. The knockdown cells were monitored for phenotypic changes over 108 hours following induction, and exhibited reduced doubling times, slowed or abnormal motility and partially or completely detached flagellum. Abnormal motility phenotypes included slow twitching or irregular beating that became more severe over time, such that by 72 hours post‐induction approximately 80% of the cells were immotile. Flagellar detachment increased over time, although less dramatically than immotility, affecting less than 60% of the population. The motility defect is seen early following induction and is evident in both detached and intact flagella and thus is consistent with the role of IC138 as a regulator of motility. Depletion of IC138 may disrupt the assembly of dynein I1 or other interacting proteins, possibly contributing to the phenotypes observed. Classification of other proteins missing in IC138 depleted cells will help to identify C. reinhardtii orthologs or lead to the discovery of novel IC138 binding partners. This identification could reveal additional components of T. brucei motility regulation and a provide basis for future research. Further knowledge of the protein interactions within dyneinI1/f may lead to a better understanding of the phenotypic changes resulting from IC138 depletion.Support or Funding InformationNIH 7R15AI101941‐02
Assessing Species‐Specific Determinants of DNA Binding in McrB Homologs
Authors: Hosford Christopher John , Johnson Virginia Mitchell , Chappie Joshua 
Published: November 2023

Antibiotic resistance poses a significant threat to human health. The lagging development of new antibiotics and the rapid exchange of resistance genes have created a need for alternative methods to combat emerging ‘superbugs’. One promising strategy involves using lytic bacteriophages (phages) – bacterial‐specific viruses that lyse and kill their hosts while releasing their progeny. Modification‐dependent restriction systems (MDRS) are innate bacterial defense systems that recognize and degrade modified phage DNA, thus preventing mature phage production in the host. MDRS are highly conserved in antibiotic resistant bacteria and a number of phage‐encoded inhibitors have been identified. Understanding the molecular mechanisms of these systems can therefore provide a means to increase the efficacy and therapeutic potential of phages.McrBC is a two‐component MDRS that restricts DNA containing 4‐, 5‐, or 5‐hydroxy‐methyl cytosines. The first component, McrB, contains an N‐terminal domain that recognizes and binds the modified site and a C‐terminal AAA+ motor domain that hydrolyzes GTP and mediates nucleotide‐dependent oligomerization. Recent crystallographic data shows that Escherichia coli (E.coli) McrB flips the methylated cytosine out of the duplex and stabilizes the resulting gap by inserting a tyrosine residue. However, this residue is not conserved suggesting that other mechanisms may exist for binding modified DNA. To examine the species‐specific determinants of McrB DNA‐binding, we have purified the putative DNA binding domains of different bacterial and archaeal McrB homologs and compared their ability to bind modified DNA with the prototypical E.coli McrB DNA‐binding domain. Thermococcus gammatolerans (T.gammatolerans) McrB binds the same DNA sequences as E. coli despite very little sequence homology. Bacillus cereus (B.cereus) McrB, in contrast, lacks a canonical N‐terminal domain and instead contains a novel domain that is inserted in the AAA+ motor core. This insert domain does not bind DNA containing methylated cytosines, suggesting it directs the conserved cleavage machinery to a different target. Furthermore, we have also crystallized the putative B.cereus and T.gammatolerans DNA binding domain and carried out preliminary X‐ray diffraction analysis. Together, these data provide insights into the substrate preferences of different McrB family members and have the potential to aid in the design of novel species‐specific therapeutics for treating multi‐drug resistant bacterial infections.
The Role of the C1q Domain of Zebrafish Otolin1a in Otolith Morphogenesis
Authors: Das Olipriya 
Published: November 2023

Otolins are constituent proteins of vertebrate otoliths and otoconia, acellular biominerals that are essential for detecting linear accelerations in the inner ear. Based on their sequence similarity to the atypical collagens VIII and X, the otolin peptides are predicted to comprise an N‐terminal signal sequence, an internal collagen domain, and a C‐terminal C1q domain. C1q domains have been shown to trimerize on their own as well as direct the trimerization of larger proteins. In the case of collagen VIII and X, the protein trimers then form a three‐dimensional matrix, with the C1q and collagen domains as the hubs and spokes, respectively. By analogy, we hypothesized that the C1q domain of otolins directs the assembly of protein trimers that are required to form the extracellular matrix of otoliths and otoconia. We tested whether the C1q domain confers trimerization on a protein that normally exists as a monomers: we investigate whether a fusion of the Otolin1a C1q domain with thioredoxin (TrxC1q) also trimerizes. The zebrafish Otolin1a C1q domain was expressed in bacteria fused to thioredoxin. Multimerization of purified recombinant fusion proteins was assessed by size‐exclusion chromatography and gel electrophoresis. Under denaturing conditions, the recombinant fusion proteins formed monomers and trimers, but not dimers or higher‐ order complexes. Under native conditions, they formed only higher‐order complexes. In contrast, thioredoxin alone appeared as a monomer. Our results are consistent with the proposed ability of the Otolin C1q domain to form trimers that, in turn, assemble into higher‐order complexes. Experiments are underway to characterize the various complexes in more detail and to test whether the C1q domain is necessary to trimerize full‐length Otolin1a.Support or Funding InformationSupported by NIH R01DC006962
Altering the Enantioselectivity of Tyrosyl‐tRNA Synthetase by Insertion of a Stereospecific Editing Domain
Authors: First Eric Allen , Richardson Charles 
Published: November 2023

Translation of mRNA by the ribosome is stereospecific, with only L‐amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of D‐amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl‐tRNA synthetases, (2) binding of aminoacyl‐tRNAs to EF‐Tu, and (3) recognition of aminoacyl‐tRNAs by the ribosome. As a first step towards incorporating D‐amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl‐tRNA synthetase. This enzyme is unusual among aminoacyl‐tRNA synthetases, as it can aminoacylate tRNA with D‐tyrosine (albeit at a 30‐fold reduced rate compared to L‐tyrosine). To change the enantioselectivity of tyrosyl‐tRNA synthetase, we introduced the post‐transfer editing domain from phenylalanyl‐tRNA synthetase into the connective polypeptide 1 domain of tyrosyl‐tRNA synthetase. We show that the phenylalanyl‐tRNA synthetase editing domain is stereospecific, hydrolyzing L‐Tyr‐tRNA, but not D‐Tyr‐tRNA. We further show that inserting the editing domain into tyrosyl‐tRNA synthetase decreases the activity of the synthetic site in tyrosyl‐tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the L‐Tyr‐tRNA product. Overall, inserting the phenylalanyl‐tRNA synthetase editing domain, results in a two‐fold shift in the enantioselectivity of tyrosyl‐tRNA synthetase towards the D‐Tyr‐tRNA product. This effect is further enhanced by introducing hyperactivating mutations into the editing domain, with one variant (F145A/S211A) preferentially aminoacylating tRNA with D‐tyrosine when equimolar concentrations of L‐ and D‐tyrosine are present.Support or Funding InformationThis research was supported by funds from Louisiana State University Health Sciences Center in Shreveport and the Biomedical Research Foundation of Northwest Louisiana.
APOBEC3F‐Vif Binding Interface Elucidated by Multiple Experimental Approaches
Authors: Harris Reuben , Richards Christopher , Shaban Nadine , Anderson Brett , Demir Ozlem , Amaro Rommie 
Published: November 2023

BackgroundAPOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV‐1 Vif protein, which subverts cellular CBF‐beta and scaffolds the assembly of a polyubiquitin ligase complex to degrade these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host‐pathogen conflict has been elusive.MethodsWe combine virus adaptation, structural, and computational studies to interrogate the APOBEC3F‐Vif interface and build a robust model for this vital host‐pathogen interaction.ResultsFirst, new structures of the Vif binding domain of APOBEC3F were solved by x‐ray crystallography. Second, a recurring compensatory amino acid substitution in HIV‐1 Vif from virus adaptation experiments provided an initial docking constraint, and microsecond molecular dynamics simulations were used to optimize interface contacts. Third, single‐cycle and spreading virus infectivity experiments were used to validate a long‐lasting electrostatic interaction predicted by molecular dynamics simulations to occur between APOBEC3F E289 and HIV‐1 Vif R15.ConclusionsOur studies have yielded a comprehensive structural model for the APOBEC3F‐Vif interaction, which explains most prior genetic data. Taken together with the dynamic nature of this host‐pathogen interaction, our data suggest a “wobble model” to explain how HIV‐1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, explain how pathogens may evolve to escape innate host defenses (i.e., a molecular explanation for the Red Queen hypothesis). Our studies also suggest that the APOBEC‐Vif interface may be a less favorable target for new antiviral drug development in comparison to other heterologous surfaces of the Vif‐CBF‐beta ubiquitin ligase complex.
The HECT Domain and RCC1‐like Domain 5/Herc5 Protein Exhibits Intrinsic Ubiquitin Ligase Activity
Authors: Ronchi Virginia P , Klein Jennifer , Streich Frederick , Haas Arthur 
Published: November 2023

HERC5 is a Type1 interferon‐induced Hect ligase having an essential role in the cellular innate immune response to viral and inflammatory signals. HERC5 is thought to be the principle ligase for conjugation of the interferon‐induced ISG15 ubiquitin‐like protein to a broad subset of cellular targets; however, HERC5 also is reported to ubiquitinate NM23B and the Hect domain forms a ubiquitin thioester intermediate with its Cys994 active site residue. The specificity of HERC5 for components of both ISG15 and ubiquitin conjugation has not been previously explored. In the current studies, HERC5‐limiting kinetic studies of 125I‐ISG15 chain formation showed cooperative sigmoidal kinetics with respect to UbcH8~ISG15, as evidenced by a linear Hill plot (nH ≈2) and a biphasic curve consistent with substrate inhibition, requiring two functionally‐ distinctive UbcH8~ISG15 binding sites (K0.5= 12 ±1 nM and kcat‐ISG15t = 1.8 ± 0.02 10−5 s−1). By exploiting the intrinsic ability of Hect ligases to form polyubiquitin chains in the absence of target proteins, we now show that recombinant HERC5 exhibits intrinsic ubiquitin ligase activity. A biochemically‐defined semiquantitative E2 screen showed that HERC5 assembled 125I‐polyubiquitin chains and displayed a broad E2 specificity. Similar to ISG15, HERC5 displayed cooperative sigmoidal kinetics (nH ≈ 2) for polyubiquitin chain assembly and showed substrate inhibition. The UbcH5 family was the cognate E2 for HERC5 with a value of K0.5 ≈ 400 nM and kcat‐ub range of 0.01 s−1. The inactive UbcH5BC85S‐ubiquitin oxyester substrate analog and UbcH5BC85A product analog were inhibitors of the ligase function. Inactive HERC5C994A and the blocked derivative Ac‐PheNH2 inhibited the ligase activity by possible affecting HERC5 oligomerization. Isotope dilution assays of 125I‐wild type polyubiquitin chain formation using unlabeled K>R point mutants at each of the seven ubiquitin lysines showed that HERC5 forms K48‐linked chains capable for targeting proteins for 26S proteasome‐dependent degradation. Together with early observations that HERC5 binds polysomes and target nascent polypeptides for ISG15 conjugation, the present studies suggest interferon induction of HERC5 might upregulate ubiquitin‐dependent antigen presentation; additionally, HERC5 is a potential bifunctional ligase functioning in both ubiquitin and ISG15 ligation pathways.Support or Funding InformationFunded by GM34009, GM47426 to A.L.H.