bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitin‐dependent and ‐independent degradation of p35 activation subunit of neuronal Cdk, Cdk5
Authors: Takasugi Toshiyuki , Minegishi Seiji , Saito Taro , Kawahara Hiroyuki , Hisanaga Shin‐ichi
Published: November 2023

Cyclin‐dependent kinase 5 (Cdk5) is a versatile protein kinase that is involved in various neuronal functions, such as the migration of newborn neurons, neurite outgrowth and synaptic regulation. In particular, dysregulation of Cdk5 activity induces neuronal dysfunction and cell death, resulting in neurodegenerative diseases including Alzheimer's disease. Cdk5 is activated by binding p35 protein and the cdk5 activity is determined by the protein amount of p35. P35 is a short half‐life protein that is degraded by proteasomes. While ubiquitination of p35 has been previously shown, the degradation mechanism of p35 is not yet understood completely. In order to identify the ubiquitination site, we mutated lysine residues in p35 to arginine. All lysine residues were mutated, but the lysine‐less p35 (p35 23R) was still degraded by proteasome, although the half‐life of p35 23R was three times longer than p35 WT. These results suggest that p35 is degraded ubiquitin‐independently, as well as dependently, by proteasome. We then investigated ubiquitin‐independent degradation of p35. Molecular mechanisms of ubiquitin‐independent degradation are largely unknown, but a number of proteins degraded ubiquitin‐independently by proteasome are increasing. An Nkx3.1 homeodomain transcription factor is such a protein to undergo ubiquitin‐independent proteasomal degradation. We found that p35 has the amino acid sequence similar to the ubiquitin‐independent degron in Nkx3.1. Nkx3.1 loses its degron activity by mutating Pro 221 to alanine in the degron sequence. P35 has Pro 247 in the C‐terminal cyclin‐fold, which may correspond to Pro 221 of Nkx3.1. We mutated Pro 247 to Ala in p35 WT and p35 23R to produce p35‐P247A and p35 23R‐P247A, respectively, and examined their degradation. P247A mutation stabilized p35. To reveal the mechanism of this ubiquitin‐independent degradation, we are now going to identify proteins, which bind p35 or its P247A mutant specifically.
Determination of protein turnover in E. coli cells during exit from persistence
Authors: Semanjski Maja , Germain Elsa , Gerdes Kenn , Macek Boris
Published: November 2023

Chronic infections that can relapse even after recurrent courses of antibiotic therapy are a major challenge in treatment of infectious diseases. In a number of bacterial pathogens, a very small subpopulation of cells, known as persisters, can become transiently tolerant to multiple antibiotics by restraining their growth and entering a dormant‐like state termed persistence. Key players in persistence are toxin‐antitoxin (TA) modules that encode a toxin, that inhibits cell growth, and antitoxin that regulates toxin activity. In E. coli, toxic activity of a Ser/Thr kinase HipA is responsible for growth inhibition and increased levels of persisters. In stress conditions, HipA phosphorylates and inhibits glutamate‐tRNA ligase (GltX) causing strong inhibition of translation. Such dormant cells are able to sense when conditions have improved, re‐initiate their growth and exit persistent state (resuscitate). Since the molecular mechanisms that govern the switch between persistence and resuscitation are largely unknown, we aimed to study early protein synthesis and turnover during resuscitation by performing a time‐resolved analysis of protein translation during the exit from persistent state. To that end, we employed pulse labeling with stable isotope‐labeled amino acids in cell culture (SILAC), in combination with high resolution mass spectrometry‐based proteomics. We enriched the low‐frequency persister cells by inducing the hipA gene, which caused immediate growth arrest, and treated the culture with a high dose of ampicillin to lyse and remove non‐persister cells. To trigger cell growth and resuscitation we induced the antitoxin‐encoding hipB gene. Resuscitating cells were pulse‐labeled with isotopically labeled lysine and harvested in regular intervals. In this experimental setup, only the newly synthesized proteins from resuscitated persister cells could incorporate lysine, enabling the quantification of the protein turnover during resuscitation. Within the first five hours after initiation of resuscitation we were able to measure SILAC label incorporation in up to 500 proteins and estimate their turnover rates. Intriguingly, most of the quantified proteins were involved in the regulation of transcription, translation and response to stress. We are currently setting up follow up experiments to investigate the role of several high‐turnover proteins in resuscitation. To our knowledge, this is the first application of a pulse‐chase SILAC‐based proteomic approach to study protein synthesis in the field of bacterial persistence. This study can largely help in understanding of the molecular basis of the exit from persistence, a state that is greatly responsible for reactivation of disease.
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 Information

This 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 Information

NSF 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.
Different phenolic compounds selectively slow down the hydrolysis rates of sucrose and isomaltose by mammalian mucosal α‐glucosidases
Authors: Lim Jongbin , Ferruzzi Mario G. , Hamaker Bruce R.
Published: November 2023

The mucosal α‐glucosidases are major enzymes which convert starch into free glucose in the small intestine. The α‐glucosidase complexes, maltase‐glucoamylase (MGAM) and sucrose‐isomaltase (SI), have two catalytic subunits located on the C‐ and N‐ termini (Ct‐ and Nt‐) of their origin proteins. Ct‐SI has the higher capacity to digest the α‐1, 2 linkage of sucrose, whereas Nt‐SI is involved in the hydrolytic activity for the α‐1, 6 linkage of isomaltose. In this study, we investigated the inhibition properties of phenolic compounds on Ct‐ and Nt‐SI using four different phenolic compounds [chlorogenic acid, (−)‐epicatechin, kaempferol, and naringenin] for the regulation of sucrose and isomaltose digestion in the small intestine. The tested phenolic compounds showed different inhibition properties on the hydrolysis activities for sucrose and isomaltose. Chlorogenic acid had the highest inhibition property on hydrolytic activity for sucrose, while kaempferol was a comparatively potent inhibitor for isomaltase activity of the α‐glucosidases. The different inhibition properties may be due to the different affinities of the tested phenolic compounds for Ct‐ and Nt‐SI. The different and selective inhibition properties of phenolics can be applied to modulate the digestion rate of sucrose‐rich foods or α‐1,6‐linked branched starch products for slow and improved glycemic response profiles with potential health benefit.
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 Information

Research 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 Information

SC3 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 Information

This 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 Information

A.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

Background

Reductive 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.

Methods

We 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.

Results

We 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.

Conclusion

This 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 Information

The 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 Information

NIH T32 ES007028

R01 GM118122
Novel Inhibitor Targeting ALDOA at Cysteine 289 and the Regulation of its C‐terminal Tail
Authors: Stancu Gabriel , Devkota Ashwini , Aleshin Alexander , De Jong Petrus , Cho Eun Jeong , Liddington Robert , Powis Garth , Dalby Kevin
Published: November 2023

Human fructose‐bisphosphate aldolases A (ALDOA) is a key glycolytic enzyme that catalyzes the conversion of fructose‐1,6‐bisphosphate to glyceraldehyde‐3‐phosphate and dihydroxyacetone phosphate. ALDOA has also been shown to play major moonlighting roles associated with cellular structure, mobility and proliferation. This aldolase isoenzyme is significantly overexpressed in a number of human tumors and is a potential oncogene, where it contributes to the Warburg effect in glycolysis. We recently identified a feed‐forward loop whereby ALDOA positively regulates the activity of hypoxia‐inducible transcription factor‐1 (HIF‐1) through AMPK, which in turn further activates ALDOA. This signaling pathway is a potential target in the irregular hypoxic environment of solid tumors. Through our University of Texas Targeted Therapeutic Drug Discovery & Development Program (TTP) we have identified a number of compounds that are able to target and inhibit aldolase enzymatic activity. Using mutational studies and high‐resolution X‐ray crystallography, we characterized a covalent inhibitor of ALDOA (TX1) which specifically targets the enzyme at Cysteine 289. Time dependent inhibition through TX1 at Cys289 leads to an approximate two‐fold decrease in the Km and kcat of ALDOA. When we treated MDA‐MB‐231 metastatic breast cancer cells with TX1 for 30 min, we observed an IC50 of 90 μM in cell lysates assayed for aldolase activity. Inhibition of ALDOA by TX1 appears to involve the complex regulation of the C‐terminal tail of the enzyme. We show that inhibition by TX1 is lessened when Cys289 is mutated to alanine and completely abrogated by removal of the C‐terminal tail. X‐ray crystallography reveals that targeting of Cys289 by TX1 significantly alters the conformation of the ALDOA C‐terminal tail with respect to the active site. This C‐term domain is important in controlling substrate specificity and enzymatic activity, and could play a major role in the moonlighting function of ALDOA. Moreover, the local environment of Cys289 makes it a prospective site for post‐translational modifications (PTMs) and an attractive target for mechanism based inhibition of ALDOA. We are working on understanding the redox and reactive oxygen species regulation of both Cys289 and the C‐terminal tail, which could shed light on the moonlighting potential of ALDOA within tumor signaling. In conclusion, we have shown that modulation of the C‐terminal domain of ALDOA through Cys289 represents a novel method of regulating its activity. Our work represents an important step in further defining ALDOA as a central signaling target in human cancer.

Support or Funding Information

This research was supported by the grant from CPRIT (RP110532‐P1).
Synthesis of Sphingosine Kinase 1 Inhibitors with Modifications of Zone 2
Authors: Nemeth Ansley , Grattan T. Christian
Published: November 2023

Sphingosine kinase 1 (SK1) is an enzyme known to catalyze the formation of sphingosine‐1‐phosphate (S1P) in the sphingolipid metabolic pathway. Within the cell, the formation of ceramide from the sphingolipid triggers apoptosis. If apoptosis does not occur, ceramide is catalyzed to form sphingosine. Following the process, SK1 then catalyzes the formation of S1P, which at high concentrations initiates cell proliferation in cancerous systems. Novel inhibitors for SK1 are needed to stop S1P from being produced. We have designed four new inhibitors based upon a known sphingosine kinase inhibitor (SKI) and evaluated the binding affinity of the enzyme for them in silico using the x‐ray crystallographic structure of human SK1 with Autodock Vina. These new inhibitors were created with chemical modifications intended to improve the oral bioavailability while improving or maintaining interactions with SK1. Modifications were made to the central pyrazole ring of the lead compound. The modifications included the substitution for a 5‐naphthylisoxazole (2A) ring, a 3‐naphthylisoxazole (2B) ring, a thiophene (2D) ring, and a furan (2G) ring. Through multiple syntheses, the final products of 2A, 2B, and 2G were successfully created after purification and analysis by 1H‐NMR. The new inhibitors are currently being evaluated via enzyme activity assay testing to determine how these modifications impact SKI relative to our lead molecule.
Modulation of pTyr signaling by small molecule inhibitors of SH2‐lipid interaction
Authors: Hu Yusi
Published: November 2023

The Src homology 2 (SH2) domain plays a pivotal role in phosphotyrosine (pTyr) signaling pathway. Our recent study showed that a majority of SH2 domains tightly bind lipids in the plasma membrane (PM) using a cationic site separate from the pTyr pocket, which is essential for their cellular protein‐protein interactions. To test the feasibility of modulating pTyr signaling activity by controlling SH2 domain‐lipid binding, we screened and optimized small molecule inhibitors for SH2 domain‐lipid binding. Small molecule inhibitors of the SH2 domain of spleen tyrosine kinase (Syk), which plays a key role in B cell signaling and has high affinity for PM, caused potent and specific inhibition of Syk signaling activities in DT40 B cells while exhibiting minimal activity against ZAP70 in Jurkat T cells.

Support or Funding Information

NIH GM68849
Inhibition Studies of Brugia malayi Dihydrofolate Reductase
Authors: Toska Dea , Goodey Nina , Gubler Ulrich
Published: November 2023

Brugia malayi is a parasitic worm, which is one of the three causes of lymphatic filariasis. Lymphatic filariasis is a parasitic disease in which the worms live and infect a patient's lymph system. Dihydrofolate reductase (DHFR) is an enzyme that catalyzes the reaction where dihydrofolic acid (DHF) is converted to tertrahydrofolic acid (THF). THF is important for synthesis of DNA. Literature studies suggest that DHFR is good drug target. If we can find a good inhibitor of B. malayi DHFR we can kill the worms and possibly find a cure for lymphatic filariasis. Current studies also suggest that antifolate compounds are inhibitors of B. malayi DHFR. Some of these compounds will be tested as potential inhibitors. In this study we are testing Aminopterin and Raltitrexed. A computational study has suggested that some green tea compounds are inhibitors of B. malayi DHFR. Green tea extract will be tested for inhibition of B. malayi DHFR. We have tested epigallocatechin gallate, epicatechin, epicatechin gallate, vitexin, and vitexcarpin to see if they are active inhibitors. If we find one that is an inhibitor, it will be studied further to determine other compounds with similar structures. We are also working on the crystallization of BmDHFR to look at the three‐dimensional structure and determine exactly what inhibitor is best for B. malayi DHFR. The goal is to find and inhibitor for B. malayi DHFR and find a cure for lymphatic filariasis.
Expression, Purification, And Characterization of W. Bancrofti DHFR and its Evaluation as a Possible Drug Target for Treating Lymphatic Filariasis
Authors: Tobias Andrew M , Gubler Ulrich A , Goodey Nina
Published: November 2023

Lymphatic filariasis is a disease caused by filarial nematodes that affects millions of individuals every year. A specific filarial nematode, W. bancrofti, is one organism that causes Lymphatic filariasis. This disease affects the lymphatic system and can cause severe swelling in the lower appendages. According to the World Health Organization, people living in Bangladesh, India, Indonesia, Myanmar, Nigeria, Nepal, the Philippines and others are at risk of infection by this neglected tropical disease. To help cure those infected, research is needed to find a drug able to inhibit the proliferation of W. bancrofti. The protein we are evaluating in order to possibly accomplish this task is WbDHFR. DHFR is an enzyme that reduces dihydrofolate into tetrahydrofolate (THF). THF and its derivatives are required for purine synthesis, making the enzyme crucial for cell proliferation. In this project, WbDHFR was expressed in LOBSTR strain E. coli and purified using a Hexa‐His tag and Ni‐NTA resin. The purified enzyme is active with a Vmax of 159 ± 9 nM/s and Km of 36 ± 4. The enzyme will be tested against several antifolate compounds as inhibitors.

Support or Funding Information

Science Honors Innovation Program at Montclair State University, and the American Society for Biochemistry and Molecular Biology
Inhibitory Effects of Substituted Pyrazoline Derivatives on Entamoeba histolytica Alcohol and Acetaldehyde Dehydrogenase (EhADH2) Activities
Authors: Hackey Meagan , Rossi Lauren , Espinosa Avelina
Published: November 2023

E. histolytica is a parasitic protist causing amebiasis in approximately fifty million people and 100,000 deaths worldwide annually. The bifunctional alcohol and acetaldehyde dehydrogenase EhADH2 enzyme is essential for E. histolytica trophozoite growth and survival as it is paramount in the glycolytic metabolic pathway. This enzyme is responsible for the conversion of acetyl co‐enzyme‐A to acetaldehyde and for the conversion of acetaldehyde to the final end product ethanol in the anaerobic pathogen. A thorough understanding of the role of this enzyme and its function will aid in the development of more effective chemical inhibitors for a better management of this deadly disease. We have generated three sets of first generation synthetic pyrazolines. Preliminary results show that three of the four series of compounds have inhibitory properties. Given the very high in vitro growth inhibition rates of these compounds and their mechanism of action, the target for these compounds seems to be EhADH2. Efficient pyrazolines will be tested for efficiency and specificity against purified EhADH2 and optimal inhibitory concentrations will be determined. This study will provide crucial and unique knowledge for more efficient chemotherapeutic targeting of ADHE enzymes. ADHE‐targeting agents could effectively treat a broad range of human diseases.
Understanding the link between antimicrobial properties of safranal and microbial ATP synthase
Authors: Liu Mason , Ahmad Zulfiqar
Published: November 2023

Background

A variety of phytochemicals with antimicrobial and antitumor properties have been identified to bind and inhibit ATP synthase. Saffron constituent safranal and its analogs are known to have antitumor, cytotoxic, and antimicrobial properties. Our lab has studied safranal and its structural analogs, thymol, cuminol, carvacrol, damascenone, 2,6,6‐trimethyl‐2‐cyclohexene‐1,4‐dione, and 4‐isoproply benzyl bromide induced‐inhibition of Escherichia coli ATP synthase and their effect on cell growth to investigate any link between antimicrobial properties of these compounds and ATP synthase inhibition.

Methods

Wild type and mutant E. coli strains were grown to late log phase. Cells were harvested by centrifugation and lysed by French Press to isolate the membrane bound F1FO ATP synthase. Null E. coli strain pUC118 with deleted ATPase gene was used as a negative control. Safranal and its analogs induced inhibitory studies were performed on membrane bound F1FO ATP synthase. Growth of wild‐type, mutant, and null strain in limiting glucose and succinate was also examined in presence and absence of safranal and its analogs.

Results

Safranal and its analogs inhibited wild‐type enzyme to variable degrees. While safranal caused 100% inhibition of wild‐type F1FO ATP synthase, only ~50% inhibition occurred for the mutant ATP synthase. Inhibitory profiles of mutant E. coli strains indicated the involvement of specific amino acids in the binding of safranal and its analogs to ATP synthase. Furthermore, safranal, thymol, cuminol, carvacrol, and damascenone fully abrogated the growth of wild‐type E. coli cells and had little or no effect on the growth of null and mutant E. coli strains.

Conclusions

Safranal and its analogs appear to bind to the polyphenol binding pocket on F1 sector of ATP synthase. The antimicrobial properties of safranal and its analogs can be linked to their binding and inhibition of ATP synthase. Thus, our results support the idea that ATP synthase can be used as a molecular drug target to combat microbial infections.

Support or Funding Information

This work was supported by the National Institutes of Health Grant GM085771 to ZA and KCOM Biomedical Sciences Graduate Program Grant Award # 850‐619 to ML and ZA.
Development of selective inhibitor of S‐adenosylmethionine synthetase in Cryptosporidium parvum
Authors: Dziobak Miranda , Parungao Gwenn , Viola Ronald
Published: November 2023

Protozoan parasites have been responsible for diseases that cause significantly high morbidity and mortality rates, as well as an economic burden. The current antiprotozoal drugs suffer from multiple liabilities related to cost, efficacy, and resistance. In this study, Cryptosporidium parvum (Cpa), the protozoan parasite that causes cryptosporidiosis – a particular concern for immunocompromised patients, was targeted for the development of an antiprotozoal drug. S‐adenosylmethionine synthetase (AdoMet synthetase ‐ metK) is the enzyme that catalyzes biosynthesis of S‐adenosylmethionine (AdoMet), an essential metabolite for all living organisms. An E. coli knockout strain (ΔmetK) used as the host for the plasmid‐containing metKCpa. Expression of this gene was shown to rescue the growth of this E. coli strain. A shorter lag phase was observed for growth in rich medium, and addition of methionine shortened the lag phase in MOPS‐glucose minimal medium. This complemented strain can now be used to screen for biologically active inhibitors against this enzyme. Furthermore, Cpa metK has been overexpressed and purified for kinetic characterization, and crystallization to study structure‐based inhibitor optimization. The results allow the further development of inhibitors for other protozoan parasites.
Inhibition of Myeloperoxidase by Staphylococcal SPIN Proteins
Authors: Ploscariu Nicoleta T. , de Jong Nienke W.M. , Ramyar Kasra X. , Garcia Brandon L. , Herrera Alvaro I. , Prakash Om , Haas Pieter‐Jan , van Strijp Jos A.G. , Geisbrecht Brian V.
Published: November 2023

Neutrophils are the most abundant type of white blood cells in humans and the most prominent cellular component of the innate immune system. They can be rapidly activated by various biochemical stimuli during the earliest phase of bacterial invasion and recruited into the sites of infection. Neutrophils contain subcellular granules that are replete with vital anti‐bacterial enzymes, including a series of chymotrypsin‐like Neutrophil Serine Proteases (NSPs) as well as a heme‐containing Myeloperoxidase (MPO). Work from the last decade has demonstrated that the pathogenic bacterium Staphylococcus aureus produces an array of virulence proteins that disrupt normal function of the human innate immune system. The combined activities of these proteins are believed to prolong bacterial survival within the host, allowing for infections to take hold. Given the critical role of neutrophils in responding to the initial stages of staphylococcal infection, we predicted that S. aureus might also express proteins that inhibit function of neutrophil granule components. Indeed, we recently identified two novel classes of S. aureus secreted proteins, EAP domain proteins and SPIN, which act as inhibitors of NSPs and MPO, respectively.

SPIN (Staphylococcal Peroxidase INhibitor) is a previously uncharacterized protein found only in Staphylococci. SPIN potently inhibits MPO activity in a number of functional assays by forming a nanomolar affinity complex with MPO. To gain insight into the structure/function relationships of SPIN, we determined its structure bound to MPO by X‐ray crystallography. The structure of this complex implicates the N‐terminal ~10 amino acids as essential for SPIN function. To identify and further characterize key residues for SPIN function in more detail, we examined three types of SPIN proteins using structural methods, direct binding assays, and functional assays for MPO activity: (i) deletion mutants, (ii) SPIN proteins originating from divergent staphylococcal species, and (iii) a synthetically‐designed SPIN protein. The differences in binding and inhibition of MPO by these proteins not only provide a route for identifying the structure/function relationships in SPIN, they also constitute a template for the design of high‐affinity, synthetic MPO inhibitors based upon the properties of this unique immune evasion protein.

Support or Funding Information

AI11120302
Biophysical Characterization and Catalytic Reactivity of Rubrerythrin and Symerythrin Model Proteins
Authors: Pellegrino Jenna , Bell Katherine A. , Polinski Rachel Z. , Cimerol Sabrina N. , Jacobs Ari , Solomon Edward I. , Reig Amanda
Published: November 2023

Rubrerythrins (Rbr) and symerythrins (Sym) are diiron carboxylate enzymes that function as both ferroxidases and peroxidases in two‐electron reduction reactions. This peroxidase activity, uniquely preferential in Rbr and Sym as compared to other members of the ferritin‐like superfamily (FLSF), is thought to result from the one or two additional carboxylate active site amino acids, compared to the FLSF archetype. The relationship between these structural features and the altered function is currently not well understood. To investigate these phenomena, a de novo‐designed protein model system was computationally designed to demonstrate the canonical 4‐helix bundle and 2His/4‐carboxylate active site structure of FLSF enzymes and to mimic their reactivities. Modifications to this model resulted in G4DFsc, which exhibits higher solubility and stability as well as increased active site accessibility. Using the G4DFsc system, Rbr‐ and Sym‐like proteins were achieved by introducing either aspartate (D) or glutamate (E) residues at active site positions 14, 43, and/or 47. The structural and catalytic properties of these systems were investigated using metal‐binding, protein‐folding, and reactivity assays at pH 7 and pH 7.5. Data show that the double mutants exhibit the weakest metal‐binding capacity at pH 7. These proteins also show slower rates of 4‐aminophenol oxidation than the original G4DFsc protein. The G47D variant shows the greatest catalytic capacity, with 4‐aminophenol oxidation rates increased 11‐fold over control reactions and 2‐fold compared to G4DFsc. These results provide insight into how particular carboxylate residues in the G4DFsc active site affect its ability to react with dioxygen and hydrogen peroxide. Preliminary cobalt titration curves show the tightest binding between the metal and G4DFsc. Interestingly, the dissociation constant of G47D is 4 times greater.

Support or Funding Information

Howard Hughes Medical Institute (HHMI), National Institutes of Health (NIH) R15GM110657
Development of Phospholipase A2 Receptor Antigen Binding Monobodies for the Inhibition of the Idiopathic Membranous Glomerulonephritis Auto‐antibody Binding
Authors: Stoddard Shana V , Zaravar AliReza , May Xavier , Goldman Liam
Published: November 2023

The use of non‐specific treatment options in autoimmune therapy today unfortunately causes patients to be prone to other infections. This non‐specific therapy suppresses the entire patient's immunes system also making it harder to fight of basic infections. Thus therapies need to be developed which can accurately target only the aberrant portion of the immune system that contributes to the autoimmune disorder (AD) specific to the individual. The work here focuses on design of antigen specific binding monobodies as a targeted means to treat AD, and specifically idiopathic membranous glomerulonephritis. Sixty monobodies were designed and optimized using in silico mutagenesis. Protein‐protein docking simulations were performed using Rosetta to evaluate for maximum binding efficiency against the ricin and the CTLD2 epitopes present on the PLA2R antigen. Our results show improved binding scores and selectivity of monobodies against the phospholipase A2 receptor (PLA2R) antigens. Initial binding energy, measured in rosetta energy units (REU), were −2.863 and −2.633 for the SH13 template and ySMB‐1 templates respectively. The M005 mutation designed in the SH13 improved binding scores to −5.32 REU. The M006 ySMB‐1 mutant showed the most in silico selectivity between the two epitope regions on the PLA2R antigen having a score of −2.966 REU against the CTLD2 epitope and a better score of −3.736 against the ricin epitope. The SH13 M005 mutant showed the greatest binding efficiency having a score of −5.319 REU. However, this mutant also binds the CTLD2 epitope (binding energy score produced was −5.32 REU), suggesting that it will not have selectivity. Several molecular interactions were identified that facilitated increased binding of the monobodies to the epitope regions compared to the original template monobodies. Molecular interactions leading to selectivity are also being evaluated. Seven monobody candidates were selected for expression which was performed in E. coli followed by subsequent purification using affinity chromatography for experimental verification of computational results is using ELISA. This work has implications for new antigen specific approach to AD therapy.

Support or Funding Information

William Randolph Hearst Foundation and Rhodes College Department of Chemistry