bluuebunny/crossref_metadata_2025_split huggingface dataset:
High throughput quantification of nucleated protein phase behavior in vivo
Authors: Halfmann Randal , Khan Tarique , Bruner Ellen , Kandola Tejbir 
Published: November 2023

Phase separation is a foundational principle for spatiotemporal regulation of protein activity. A subset of phase separating proteins, known as prions, produce switch‐like, irreversible changes in protein activity that effectively decide the fates of cells and those of their progeny. For some proteins, these switches are physiological, as we have recently shown for certain innate immune signaling proteins. But for others, they are clearly pathological, as for Alzheimer's and other degenerative disease‐associated proteins. What physical properties distinguish physiological from pathological prions, and how does prion formation differ from that of other condensed protein phases such as liquid droplets? In order to decipher the rules that govern prion‐like phase transitions and explore the breadth of their biological effects, we developed a powerful cell‐based fluorescence assay termed Amphifluoric FRET (AmFRET). AmFRET combines the use of photoconvertible fluorescent proteins with a high‐variance protein expression system and imaging flow cytometry to enable precise, high‐throughput mapping of the concentration‐dependencies and kinetics of protein self‐assembly. We have used AmFRET to characterize phase behaviors for hundreds of known and putative prion‐forming proteins from two extremes of conformational space: 1) intrinsically disordered regions commonly involved in gene regulation, and 2) well‐folded death domains from mammalian signaling pathways. Our findings enable us to derive a quantitative physical description of “prionness”, and reveal an unprecedented prevalence of prion behavior in cell signaling pathways that execute terminal cell‐fate decisions. In addition, we have discovered a multiplicity of nucleated phases accessible to individual proteins; the phases differ in their thermodynamic properties and biological consequences.Support or Funding InformationThis work is funded by an NIH Director's Early Independence Award (DP5OD009152) and the Stowers Institute for Medical Research.
Remodeling the Actin Cytoskeleton by Ubiquitin‐Dependent Proteolysis
Authors: Mannix Katelynn M. , Gerdes Juli , Hudson Andrew , Kaufman Ronit , Cooley Lynn 
Published: November 2023

In order for cells to perform their various functions, they must assemble and continually regulate specialized actin‐based structures and networks. Remodeling of actin networks is critical to allow cells to respond to external stimuli and carry out important processes during development. Ubiquitin‐mediated protein degradation is emerging as a new regulatory mode of cytoskeletal organization and remodeling. The Drosophila ring canal provides a useful model system to study how ubiquitination affects the actin cytoskeleton. Ring canals are intercellular bridges that grow during development from 1 μm to 10 μm in diameter as a result of a dynamic and robust actin cytoskeleton. Interestingly, mutation of kelch results in aberrant accumulation of F‐actin that completely occludes the lumen of ring canals (referred to as the kelch‐like phenotype). We have recently characterized Kelch function as the substrate adaptor component of a Cullin3‐based ubiquitin ligase complex (notated as CRL3Kelch). We showed that the CRL3Kelch ubiquitin ligase functions to target a ring canal substrate for ubiquitination and proteasomal degradation – a process essential for the proper regulation of the ring canal actin cytoskeleton. We are currently investigating HtsRC, a novel ring canal protein, as the substrate based on the following genetic evidence: (1) overexpression of HtsRC induces kelch‐like RCs; (2) downregulation of HtsRC suppresses the kelch‐like phenotype; and (3) HtsRC protein levels are dependent on Kelch protein levels. We are currently performing biochemical assays to validate HtsRC as the substrate. Preliminary data from cultured Drosophila S2 cells suggests that HtsRC is ubiquitinated and degraded by the proteasome, and its ubiquitination may be dependent on CRL3Kelch. Additionally, we are performing co‐IP binding assays to map the HtsRC‐Kelch binding interaction. Given the role HtsRC may be playing in the crucial remodeling and organization of the RC actin cytoskeleton during development, we are now interested in identifying its binding partners. To this end, we fused HtsRC to the APEX enzyme to achieve proximity‐dependent biotinylation of HtsRC‐interacting proteins in egg chambers. These biotinylated proteins can be captured by streptavidin beads and identified by mass spectrometry (MS). Excitingly, HtsRC::APEX MS hits include known ring canal proteins and are unique compared to Pav::APEX (another RC‐specific fusion) hits, indicating that this technique can be used to identify specific interacting proteins with high spatial specificity in living tissue.Support or Funding InformationThis work was funded by NIH RO1 GM043301 grant to L.C.K.M.M. was supported in part by the National Institute of General Medical Sciences NIH training grant T32 GM007223.
Conformational Locking and Unlocking in the Ubiquitin Family of Proteins
Authors: Kelly Ryan Taylor , Berndsen Christopher 
Published: November 2023

Ubiquitin fold modifier 1 (UFM1) is an ubiquitin‐like protein (UBL) found in eukaryotic organisms which plays a crucial role in cell cycle regulation, signal transduction, and more. A three step enzymatic pathway composed of enzymes E1, E2, and E3 is utilized to attach UFM1 to its target protein. This conjugation pathway is common to Ubiquitin and other UBL proteins such as UFM1, ATG8, and SUMO. The crystal structure of UFM1 and its E1 (UBA5) in complex shows that UFM1 binds to the adenylation domain of UBA5 and interacts with a separate ubiquitin interacting sequence (UIS) in the C‐terminus of UBA5. The UIS interacts with UFM1 on the opposite side of the UBL protein from the adenylation domain. The reason for this second interaction site is unclear. Through molecular dynamics simulations paired with ConSurf and STRESS analysis, we analyzed UFM1 bound to the UIS sequence in the absence of the UBA5 adenylation domain. The residues in the adenylation interaction site of UFM1 have less movement when the UIS peptide was bound to UFM1 and formed a structure that aligns well UFM1 bound to the UBA5 adenylation domain. These data suggest the UIS induces formation of the proper structure for interacting with UBA5 and may enhance the affinity by locking the conformation of UFM1. The “locked” conformation of UFM1 is observed in the absence of the peptide, but less frequently. Through additional simulations on Ubiquitin and Ubiquitin‐like proteins, we show that similar allosteric trends exist for ATG8 which like UFM1 uses a non‐canonical E1 enzyme while Ubiquitin and SUMO do not show this behavior. Ubiquitin and SUMO, instead show increased motion (“unlocking”) upon binding to known interacting partners. Non‐canonical E1 enzymes lack of some substrate binding domains found in the canonical E1 enzymes suggesting this locking mechanism may compensate for the lack of these domains.Support or Funding InformationNSF REU (CHE‐1461175)US Israel Binational Science Foundation
ScMC4 and ScMC5 are Metacaspases Active in Cleaving Peptides after Arg Residues
Authors: McDonald Molly , Fox Kristin 
Published: November 2023

Apoptosis is a highly organized form of programmed cell death (PCD) that can be initiated as part of development in higher eukaryotes, or in response to environmental stresses, toxic metabolites, and nutrient starvation. Caspases are well studied proteases that play a crucial role in activating PCD, and are found in all kingdoms of life. Metacaspases are caspase‐related proteases that are found in eukaryotes that do not have any caspases, such as plants and fungi. Though both metacaspases and caspases contain a catalytic cysteine‐histidine dyad, metacaspases cleave proteins after a lysine or arginine residue while caspases cleave after an aspartate residue. A subset of metacaspases contain an N‐terminal prodomain that may undergo autocatalysis, and is thought to be required for activity. Five metacaspase genes (ScMC1‐5) have been identified in the complete genome of Schizophyllum commune, a split gilled fungus that is a reported human pathogen. ScMC1 and ScMC3 have been previously characterized, and the genes for ScMC5 both with and without the prodomain (ScMC5Δpro) have been inserted into plasmids for expression.This project focuses on the expression, purification, and characterization of ScMC4 and ScMC5, as well as the development of protocols for determining enzyme concentration. The genes for ScMC4 and ScMC4Δpro were isolated using PCR and were successfully inserted into a plasmid fused with the small ubiquitin modifier (SUMO) gene using Gibson Assembly. Both ScMC4 and ScMC5 with and without the prodomain were expressed and showed activity and kinetic constants comparable to other metacaspases. The activity assay used to measure protein activity utilizes the substrate Z‐GRR‐AMC. To quantify the amount of cleavage, a standard curve of AMC fluorescence relative to concentration was created. A protocol for quantifying the amount of active metacaspase was developed by titrating the active site with the suicide inhibitor valine‐arginine‐proline‐arginine‐fluoromethylketone (VRPR‐FMK). Through these studies, S. commune metacaspases can be further characterized and a more in depth understanding of metacaspase activity can be utilized for the development of antifungal medications.
Chemical Modification of Trypsin Improves Protein Digestion Efficiency
Authors: Washer Benjamin , Poe Derrick , O'Grady John , Meyer Kevin 
Published: November 2023

Proteins are molecules of great interest, since their prevalence and diversity of functionality are so profound. Due to this, proteins are widely used as catalysts in biological reactions, used as probes in biological systems, etc. One example, trypsin, a well characterized serine protease, is a protein used in the cleaving or “digestion” of proteins for use in mass spectrometry and structural analysis.However, like other proteins, trypsin often functions only under a very limited range of conditions and experiences a reduction in activity outside of this range. In collaboration with Perfinity Biosciences, experiments focusing on the chemical modification of trypsin with the intention of improving the enzymatic activity and digestion efficiency of trypsin under various conditions. Modification experiments were tested by varying conditions such as buffer, temperature, thermal stress and matrix complexity under which the modified trypsin was used to digest both a simple protein (native human insulin) and a more complex antibody (Human immunoglobin G).Results indicate that the modified trypsin performs better than the unmodified trypsin under standard conditions and also maintains a relatively high level of activity under other, more stressful conditions.
Determining ClpXP Substrates Under Different Nutrient Conditions in Escherichia coli
Authors: Peterson Celeste , Mendes Amberely , Lambert Adrian , Papaj Agllai , Ngo Pauline , Von Rosen Tatjana 
Published: November 2023

Cellular protein concentrations reflect a balance between synthesis, degradation, and dilution by cell growth. Synthesis is routinely analyzed in high‐throughput experiments, but fewer studies consider degradation, particularly under non‐optimal growth conditions such as starvation. ClpXP is a central and conserved protease that degrades numerous proteins. Here, we modified the ClpXP protease trap assay to identify which proteins are degraded under glucose and no glucose conditions. Substrates involved in replication, protein synthesis, and metabolism were found to be associated with ClpXP during starvation. Conversely, substrates involved in the stress response were more likely to be degraded in glucose conditions. Almost all of these proteins had a canonical C or N terminal ClpX tag on them. Thus, the ClpXP protease markedly remodels its substrate profile during starvation.Support or Funding InformationResearch Corporation for Scientific Advancement
Biochemical Studies of Recombinant AaCHYMO from the Female Aedes Aegypti Mosquito
Authors: Burata Olive , Rascon Alberto A. 
Published: November 2023

The new emergence of the Zika arbovirus within the Americas has brought great attention to the female Aedes aegypti mosquito. This anthropophilic mosquito undergoes multiple blood feedings throughout its lifetime in order to successfully provide nutrients for the gonotrophic cycle, the process of egg laying and oocyte maturation. In doing so, the Ae. aegypti has conveniently become the dominant vector in transmitting Zika and various other arboviruses. Zika virus infection has already been linked to microcephaly in newborn babies, an abnormal development of the brain characterized by a significantly smaller than average head size. Current defenses against these mosquitos mainly involve an exaggerated usage of pesticides that have led to an increase in pesticide‐resistant mosquitos, as well as collateral injury towards pollinators. Since midgut serine proteases within the Ae. aegypti are required for digestion of blood meal proteins into the nutrients required to fuel the gonotrophic cycle, inhibiting these enzymes may provide a more specific method of population control.Blood meal digestion has been shown to have an early and a late phase, with the majority of blood meal digestion and trypsin‐like activity being observed in the late phase. However, little is known about a different class of serine proteases, the chymotrypsin‐like serine proteases. One chymotrypsin‐like enzyme, AaCHYMO, was previously reported to be expressed in the early phase of midgut digestion, but no biochemical information is available and its role in the blood meal digestion process is unknown. Therefore, a recombinant form of AaCHYMO was constructed using the bacterial expression pET28a vector and expressed using Shuffle T7 E. coli cells. Purification of the recombinant form of AaCHYMO was successful, but surprisingly the enzyme isolated was the active form, potentially indicating the possibility of this chymotrypsin to be auto‐catalytic.Support or Funding InformationSC3 Research Grant from NIH
Characterization of the Nicotinic Acid and 6‐Hydroxynicotinic Acid Dehydrogenase Complexes in Bacillus niacini
Authors: Kalafatis Kathleen Irina , Snider Mark J. 
Published: November 2023

N‐Heteroaromatic compounds (NHACs) are prominent environmental contaminants that have cytotoxic, mutagenic, and carcinogenic effects on organisms. NHACs are highly persistent within the environment and strategies to remove these contaminants from aquatic environments have been largely ineffective. Therefore, more research regarding bioremediation is warranted. Several bacteria have been discovered to degrade NHACs, such as Pseudomonas, Bordetella, and Bacillus. These organisms can also catabolize nicotinic acid providing a model NHAC pathway to investigate. The nicotinic acid degradation mechanisms in P. putida and B. pertussis have largely been determined. The pathway of nicotinic acid catabolism and the catalysts in B. niacini are not established. Elucidating this mechanism will allow for optimizing bioremediation applications. Interestingly, all three organisms share a common first hydroxylation step catalyzed by nicotinic acid dehydrogenase. The pathway in B. niacini then diverges with a second hydroxylation step catalyzed by 6‐hydroxynicotinic acid dehydrogenase. Through genome sequencing, a novel gene cluster has been identified, putatively suggesting that four proteins are involved in the two‐hydroxylation steps: NicA1, NicA‐2, NicB1, and NicB2. It is further hypothesized that these proteins form multi‐subunit complexes known as NicAB, perhaps similar to the nicotinate dehydrogenase complex in Eubacterium barkeri. To confirm the gene annotations and determine the oligomerization of the functional NicAB complex in B. niacini, specific deletion mutants will be constructed and analyzed for their ability to survive on minimal media supplemented with nicotinic acid or 6‐hydroxynicotinic acid.Support or Funding InformationThis work was funded by the Department of Chemistry and the Henry J. Copeland Fund for Independent Study at The College of Wooster.
Engineering Nanobioactive Hydrogels for Stem cells and Regenerative Medicine
Authors: Paul Arghya 
Published: November 2023

Nanotechnology and microscale research work to control stem cell microenvironment has led to its diverse applications in the field of biomedicine, particularly to study cell‐biomaterial interactions, tissue engineering and stem cell based regenerative medicine. Interestingly, a wide variety of biomimetic nanomaterials are in studies to mimic the native microarchitecture and biological cues of natural matrices. Such bioactive nanomaterials can be used as biotransporters for therapeutic applications. They can also be used to fabricate drug carrying cardiovascular devices and cardiac implants. The talk will address the emerging micro and nanoscale technologies to fabricate such bioactive materials for biomedical applications and provide a brief overview of his current research goals towards this direction at his Biointel Laboratory.Support or Funding InformationA.P. acknowledges an investigator grant provided by the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the NIH Award Number P20GM103638. He also acknowledges the University of Kansas New Faculty General Research Fund.
Heterologous Production of Functional Chloroform Reductive Dehalogenase
Authors: Jugder Bat‐Erdene , Payne Karl A. P. , Fisher Karl , Manefield Michael , Lee Matthew , Leys David , Marquis Christopher P. 
Published: November 2023

BackgroundReductive dehalogenases (RDase) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). RDases are membrane‐associated proteins whose activity is dependent on protein‐associated cobalamin and iron‐sulfur clusters. The heterologous expression of respiratory RDases is reported to be extremely challenging. Dehalobacter strain UNSWDHB is an ORB capable of respiring chloroform (CF, or trichloromethane) as an electron acceptor for the generation of cellular energy and as a consequence, dechlorinates CF to dichloromethane. TmrA enzyme is responsible for this reaction (i.e. CF‐RDase) and has been purified from Dehalobacter strain UNSWDHB and partly characterized. This study explores heterologous expression of the same enzyme.MethodsWe cloned a codon optimized tmrA with an N‐terminal hexa‐His‐tag replacing the TAT signal sequence and potential transmembrane helix region into the pPT7 plasmid. The pPT7‐tmrA plasmid was transformed into Bacillus megaterium MS941 containing the pT7‐RNAP plasmid. Xylose‐inducible expression of tmrA was performed aerobically in TB media at 17°C. The cells were disrupted anaerobically using a French press. CF dechlorination activity of the soluble protein fraction was measured by gas chromatography using Ti(III)citrate and methyl viologen as electron donors.ResultsWe expressed the N‐His tagged TmrA protein in B. megaterium cells. Soluble protein fractions exhibited CF dechlorination activity in vitro. Further biochemical characterizations will be undertaken on the purified enzyme.ConclusionThis is the first report of heterologous expression of soluble and active respiratory RDase in B. megaterium. The CF reducing TmrA enzyme was expressed in cobalamin‐producing B. megaterium cells, and dichloromethane production could be demonstrated in the soluble protein fractions.Support or Funding InformationThe authors wish to acknowledge the University of New SouthWales (UNSW) for providing scholarship support for BJ.
Oxidative Inhibition of the ω‐Hydroxylation Activity of Cytochrome P450 4A11 by Sulfenylation of the Heme‐Thiolate Cysteine
Authors: Albertolle Matthew Edward , Kim Donghak , Guengerich F. Peter 
Published: November 2023

Cytochrome P450 (CYP) 4A11 is an ω‐hydroxylase that catalyzes the oxidation of arachidonic acid to 20‐hydroxyeicosatetraenoic acid (20‐HETE). 20‐HETE can be pro‐hypertensive by constricting microvasculature or anti‐hypertensive by inhibiting salt reabsorption in the kidney. Thus, 20‐HETE and CYP4A11 have important roles in blood pressure regulation. Using isotope‐coded affinity tag (ICAT) technology, cysteine‐sulfenic acids were alkylated by d6‐dimedone followed by counter‐alkylation of free thiolates by d0‐iododimedone. Peptides were analyzed by nLC‐MS/MS and using the software, Skyline, peak integrals of heavy/light peptides were then compared to obtain a ratio of thiol sulfenylation. Loss/recovery of the heme‐thiolate ligand was monitored spectrophotometrically in anaerobic conditions using first P450 oxidoreductase and then dithionite as iron and thiol reducing agents, respectively. The ratio of sulfenic acid/thiolate of the heme ligand cysteine was correlated with the inhibition of enzymatic activity by H2O2 in a time‐ and concentration‐dependent manner. This loss and subsequent recovery of the heme‐thiolate ligand were observed spectrophotometrically. 20‐HETE production has been implicated in the development of hypertension in mice and correlates with vascular diseases in humans. Perturbations of the vascular system are known to induce oxidative signaling and oxidative stress. This redox switch in CYP4A11 may be part of a control mechanism to limit 20‐HETE production in vivo.Support or Funding InformationNIH T32 ES007028R01 GM118122