bluuebunny/crossref_metadata_2025_split huggingface dataset:
A nucleotide‐dependent switch in proteasome assembly mediated by the Nas6 chaperone
Authors: Park Soyeon , Li Frances , Sokolova Vladyslava 
Published: June 2021

The proteasome is a molecular machine essential for protein degradation. The proteasome is assembled via the 9‐subunit lid, 9‐subunit base, and 28‐subunit core particle (CP). Previous work has shown that the chaperones Rpn14, Nas6, Hsm3, and Nas2 each bind a specific ATPase subunit of the base, and antagonize base‐CP interaction. Here we show that the Nas6 chaperone also obstructs base‐lid association. Nas6 alternates between these two inhibitory modes according to the nucleotide state of the base. When ATP cannot be hydrolyzed, Nas6 interferes with base‐lid, but not base‐CP association. In contrast, under conditions of ATP hydrolysis, Nas6 obstructs base‐CP, but not base‐lid association. Modeling of Nas6 into cryo‐EM structures of the proteasome suggests that Nas6 controls both base‐lid affinity and base‐CP affinity through steric hindrance; Nas6 clashes with the lid in the ATP‐hydrolysis‐blocked proteasome, but clashes instead with the CP in the ATP‐hydrolysis‐competent proteasome. Thus, Nas6 provides a dual mechanism to control assembly at both major interfaces of the proteasome.Support or Funding InformationBoettcher Webb‐Waring Biomedical Research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relevance of the Non‐canonical Complex Formed by Proteasome Subunit α4
Authors: Panfair Dilrajkaur , Hammack Lindsay , Hopf Brett , Kusmierczyk Andrew R. 
Published: June 2021

The 20S proteasome plays an essential role in maintaining protein homeostasis as part of the larger 26S proteasome complex. It is composed of seven distinct α subunits and seven distinct β subunits arranged as a stack of four heptameric rings in an α7β7β7α7 manner. Assembly of these 28 subunits is very efficient and aided by assembly chaperones that function, in part, to prevent off‐pathway interactions. Certain α‐subunits of the proteasome form non‐canonical ring complexes in vitro. The significance of such complexes is not known but they could simply represent dead‐end, or off‐pathway, assemblies. We recently provided the first in vivo evidence of such a non‐canonical ring complex formed by the yeast α4 subunit. Levels of this complex increase when proteasome function is compromised. Here, we aim to further investigate the relevance of this α4 high molecular weight complex (α4HMWC). Using a salt bridge disruption strategy, we achieved destabilization of the α4HMWC without affecting proteasome assembly. If the α4 HMWCs are important physiologically, we may observe growth phenotypes of the resulting yeast mutant lacking this complex. Our results will help determine whether this complex is an off‐pathway, or dead‐end, product or whether it may have an independent function within the cell.Support or Funding InformationResearch Support Funds Grant from Indiana University‐Purdue University Indianapolis to A.R.KThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the Role of the L/I Zipper in the Stability of Paramyxo‐ and Pneumovirus F Proteins
Authors: Branttie Jean Mawuena , Webb Stacy R. , Dutch Rebecca E. 
Published: June 2021

Paramyxoviruses such as Hendra virus (HeV) and Measles virus (MeV) are important pathogens of worldwide significance. Others such as Parainfluenza virus (PIV5) are widely used as paramyxovirus model systems. Despite their prevalence, currently, no FDA‐approved vaccines or antiviral therapeutics are available against them. Paramyxoviruses consist of a single‐stranded negative sense RNA genome enveloped in a host‐derive membrane. Paramyxovirus glycoproteins play an important role in infectivity: the attachment protein (G, HN, or H) docks the viral particle to the host cell, and the fusion protein, (F), drives fusion with a host cell membrane, which allows the release of viral contents into the host cell. To promote successful fusion, the F protein undergoes dramatic, essentially irreversible conformational changes which must be carefully timed. To avoid unproductive triggering events, F is held in a metastable prefusion conformation until the right signal for fusion occurs. Previous studies in HeV F have shown that the transmembrane (TM) domain is important for prefusion stability with a leucine/isoleucine (L/I) zipper playing a key role. Furthermore, similar L/I zipper motifs are present across the paramyxovirus and the closely related pneumovirus families. To define the significance of the L/I zipper motifs in other paramyxo‐ and pneumovirus F proteins, we created a PIV5 F mutant protein which has alanine substitutions to the L/I zipper in the TM domain. Our preliminary data suggest that the mutant expresses; however, compared to wild type, which demonstrates a strong localization to the plasma membrane, the mutant shows a ubiquitous distribution in cells. This suggests that the mutant undergoes different trafficking events compared to the wild type. Further studies will be performed to quantify surface and total expression of the mutant, and also examine its stability and fusogenic activity. The effect of the L/I zipper will also be investigated in the context of Human metapneumovirus, a pneumovirus in order to determine whether the TM domain of paramyxo‐ and pneumoviruses present a broad therapeutic target.Support or Funding InformationWe gratefully acknowledge the NIH/NIAID for supporting our research grant R01 AI051517This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allosteric Inhibitor of ERAP1 Acts by Stabilizing a Closed Conformation
Authors: Maben Zachary , Arya Richa , Stern Lawrence 
Published: June 2021

Endoplasmic‐reticulum associated aminopeptidase 1 (ERAP1) is a M1 family zinc aminopeptidase that generates peptides for class I major histocompatibility complex (MHC‐I) presentation and T cell surveillance. ERAP1 has been crystalized in open and closed states, and is postulated to convert between them during its catalytic cycle. Small‐angle X‐ray scattering (SAXS) studies show that in solution ERAP1 predominately adopts the open conformation. A highly specific small‐molecule inhibitor of ERAP1 causes it to adopt a closed conformation, suggesting an allosteric inhibition mechanism. The compound inhibits ERAP1 peptide processing activity but activates hydrolysis activity of a dipeptide analog, leucine‐7‐amido‐4‐methylcoumarin, supporting an allosteric mechanism. The inhibitor also differentially alters enzymatic behavior of naturally occurring ERAP1 variants with lysine or arginine at position 528, which differ in their solution conformational equilbrium. Arg/Lys 528 forms an electrostatic interaction in the closed conformation that is broken when ERAP1 adopts the open conformation; computational studies predict that substitution of arginine for lysine decreases the electrostatic potential by 1.67 kT/e. We propose that this polymorphism alters the stability of the closed conformation, and that the differential behavior of the inhibitor for the ERAP1 variants is directly related to the stability of the closed conformation. The role of conformation in the catalytic cycle may be a feature of the entire M1 aminopeptidase family.Support or Funding InformationThis research was supported by NIH National Institute of Allergy and Infectious Diseases (NIAID) under the National Institutes of Health award number R01AI038996.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Glucosome: A Metabolic Compartment for Glucose Metabolism in Living Cells
Authors: Jeon Miji , Kohnhorst Casey , Kyoung Minjoung , An Songon 
Published: June 2021

Glycolytic enzymes have been suggested to form multienzyme complexes in a variety of organisms. Recently, we have shown that liver‐type phosphofructokinase 1 (PFKL) tagged with a fluorescent protein forms various sizes of cytoplasmic enzyme clusters in human cells. We also found that these PFKL clusters colocalize with other cytoplasmic rate‐limiting enzymes in glucose metabolism, supporting the spatiotemporal formation of a multienzyme complex, namely the “glucosome.” Importantly, quantitative high‐content imaging data unveil that the various sized cytoplasmic enzyme clusters play a functionally different role by shunting glucose flux into the pentose phosphate pathway or serine biosynthesis. We propose that the differing sizes of glucosome clusters represent various metabolic functions to guide the direction of glucose‐mediated carbon flux in the cell. Therefore, we envision that our study will advance the understanding of how the multienzyme metabolic assemblies are spatially compartmentalized in the cytoplasm to functionally contribute to regulate glucose flux in cells.Support or Funding InformationNational Institutes of Health Grant T32GM066706This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Organophosphate (OP) Conjugation on Structure and Dynamics of Human Acetylcholinesterase
Authors: Fajer Mikolai , Cheng Xiaolin , Kovalevsky Andrey , Gerlits Oksana , Blumenthal Donald , Taylor Palmer , Radić Zoran 
Published: June 2021

Organophosphate (OP) conjugation of the human acetylcholinesterase (hAChE) active site leads to a deadly increase of the neurotransmitter acetylcholine in the nervous system. The slow reactivation rate of OP‐hAChE by current antidotes is a major problem in effective treatment. This is a possible consequence of sterically restricted antidote access to the conjugated phosphorus in already narrow active center gorge of hAChE emphasized by available static X‐ray structural snapshots. We have conducted inelastic neutron scattering (INS) experiments on both hAChE and paraoxon (POX) conjugated hAChE (POX‐hAChE) to better understand the influence of OP conjugation on hAChE dynamics and reactivation rate limitation. The measured dynamic structure factor S(q,ω) showed a statistically significant difference at high Q between the two samples. The corresponding vibrational density of states (DOS) indicated that the difference was a result of low frequency vibrations in the 30 to 50 cm−1 range. In order to identify the particular mode of motion that is affected by OP conjugation, we compared the three X‐ray structures of the POX‐conjugated hAChE with the apo‐hAChE, and found tertiary conformation deviations in three regions: the acyl pocket, C‐terminal helix bundle and the Ω loop. We then constructed computational models of the apo‐hAChE and POX‐hAChE powder samples each with eight randomly oriented monomers. After equilibration, multiple 10 ps simulations were performed to compute the vibrational DOS via the velocity autocorrelation functions of the protein heavy atoms. An additional set of 10 ps simulations were given an impulse to each of the three specific regions. These simulations confirmed that a motion closing the acyl loop gorge was in line with experimental INS data. This motion was further shown to be coupled with the helix bundle dynamics, providing an explanation for the change in hAChE quaternary structure after OP‐conjugation observed in the SAXS data. Taken together, our results reveal detailed molecular insights into how OP‐conjugation changes the dynamic behavior of hAChEs.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering yeast endosymbionts as a first step towards laboratory evolution of mitochondria
Authors: Mehta Angad , Supekova Lubica , Supek Frantisek , Schultz Peter 
Published: June 2021

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in a protoeukaryotic cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between E. coli and the budding yeast S. cerevisiae. Fusion of yeast with E. coli ectopically expressing several genes from unrelated, intracellular bacteria was key for establishing endosymbiosis. ADP/ATP translocase‐expressing E. coli provided an energy source for a respiration‐deficient cox2 yeast mutant, enabling growth of yeast‐E. coli chimera on a non‐fermentable carbon source. Similarly, yeast provided a source of thiamin or NAD to an E. coli thiamin or NAD auxotroph respectively. Expression of several SNARE‐like protein on the surface of E. coli was also required to prevent lysosomal degradation. The engineered yeast‐E. coli chimeras sustained growth on selection medium in containing the antibiotic carbenicillin indicating the presence of intracellular E. coli which supports the growth by ATP synthesis on selection medium. Further, sf‐gfp expressing E. coli endosymbionts could be observed in the yeast by super resolution fluorescence microscopy after more than 40 doublings. This readily manipulated system should allow us to experimentally delineate host‐endosymbiont adaptations that occurred during evolution of the current much reduced mitochondrial genomeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Light Triggered Isoelectric Point and Solubility Shifts Applied to Photoactivated Insulin Depots
Authors: Nadendla Karthik , Friedman Simon H. 
Published: June 2021

We previously described photoactivated insulin depot, a minimally invasive technique of insulin delivery (1). In this approach, an inert photoactivated depot is injected under the skin. Exposure of the depot to light triggers the release of native insulin into the blood. First‐generation materials were designed to have insulin chemically attached to an insoluble polymer (for insolubility) via photocleavable linker. Though this material is effective in studying the approach in vitro and in vivo (2), it has multiple problems associated with the insoluble polymer. About 95% of the depot consisted of the non‐therapeutic polymer, which is non‐biodegradable and difficult to inject into an animal.In this work, we solved these problems by eliminating the polymer and reacting insulin with a photocleavable group containing positively charged groups. The result is an insulin with an altered pI and thus, with a different solubility (3). We achieved the insulin's insolubility by shifting its pI to physiological pH. We observed an increase 0.65 units of pI of insulin with the addition of a single positive charge. We raised the pI of insulin from 5.4 to 7.2, specifically by the addition of two positively charged groups and quenching one negative charge (carboxylate) on insulin. This material has high solubility in acidic pH, and hence can be a soluble formulation. But upon injection at skin pH, it is observed to precipitate as a depot. Upon exposure to light, the positively charged groups are removed to generate the native insulin, which can then be absorbed into the blood due to its high solubility. These materials have high insulin density (91% w/w), are soluble in acidic formulations and upon photolysis, will result in charged, soluble and excretable by‐products without leaving any traces of depot at the site of injection.Support or Funding InformationNIH‐1DP3DK106921‐01, University of Missouri Fast Track Award and UMKC School of Pharmacy Dean's Bridge FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing an Efficient Method for the Incorporation of a Series of Fluorotyrosines in Peptides via Solid Phase Peptide Synthesis
Authors: Chau Noel , Colla Alexander , Schwans Jason 
Published: June 2021

The incorporation of unnatural amino acids in peptides and proteins has furthered the investigation of various protein structures and enzyme function. Enzyme residues suggested to be important for catalysis are readily mutated and measured for energetic effects. However, the limited repertoire of naturally occurring amino acids constrains the available substitutions. To obtain a deeper understanding of how enzymes work, unnatural amino acids are used to systematically perturb enzymatic residues. Previous studies in the enzyme ketosteroid isomerase (KSI) used a series of fluorotyrosine analogs to perturb the tyrosine hydrogen bond donor. However, challenges in the synthesis of fluorotyrosine analogs limited their use. To overcome these challenges and extend the series of fluorotyrosines used in enzymatic studies, we are developing an approach to selectively incorporate fluorotyrosines in peptides using silyl‐based and fluorenylmethyloxycarbonyl (Fmoc) based chemistry. Introduction of Fmoc had moderate yields of up to 70%, while silylation of Fmoc‐tyrosine led to low yields of ~20%. Low yields may be attributed to presence of additional undesired silylation products. To overcome this limitation, a series of silylation conditions and additional protecting groups on the amino acid are being evaluated. After optimizing reaction conditions, the dually protected fluorotyrosines will then be used to generate synthetic peptides via solid phase peptide synthesis.Support or Funding InformationThis research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 5UL1GM118979‐03; 5TL4GM118980‐03; 5RL5GM118978‐03. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Inhibitors of P‐Glycoprotein from In Silico Optimizations of Inhibitor SMU‐29 using the ChemGen Program
Authors: Ammerman Lauren E. , Nanayakkara Amila K. , Aljowni Maha , Lippert Alexander R. , Vogel Pia D. , Wise John G. 
Published: June 2021

P‐glycoprotein (P‐gp) is an integral membrane efflux pump that is capable of lowering the intracellular concentration of many drugs to sub‐therapeutic levels. Consequently, P‐gp has been implicated in multidrug resistances (MDR) to cancer chemotherapies. One promising P‐gp inhibitor, SMU‐29, was previously identified by us using in silico methods (Brewer et al., Molecular Pharmacology 86, 716–726, 2014) and was later confirmed to reverse MDR in cancer cell culture experiments (Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). We created millions of SMU‐29 variants in silico using our novel medicinal chemistry program, ChemGen, in efforts to pharmacologically optimize SMU‐29. In one set of chemical variants, only part of the structure of SMU‐29 was varied; in a second set of variants, only the central structural scaffold of SMU‐29 was conserved. We used in silico docking to identify chemical variants of SMU‐29 that exhibited high estimated affinities for P‐gp's nucleotide binding domains (NBDs), and low estimated affinity for P‐gp's transmembrane drug‐binding domain (DBD). Using this approach, we identified over 2000 ChemGen‐created variants with equivalent or better estimated affinities at the P‐gp NBDs relative to SMU‐29. We selected a small set of these SMU‐29 variants for chemical synthesis and further screening in vitro against multidrug resistant cancer cell lines. All of the tested and synthesized SMU‐29 variants were observed to act as P‐gp inhibitors with improved PC50 values. Some of these variants were also observed to inhibit the breast cancer resistance protein (BCRP), a related ABC‐transporter that is also implicated in MDR in cancer. Our previous screens that identified SMU‐29 as a hit compound had a 33% P‐gp inhibitor identification success rate (hit rate); the present in silico optimization of SMU‐29 using ChemGen has demonstrated a very high success rate of identifying P‐gp inhibitors with improved affinities for the target protein.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In‐vitro compositional investigations of antioxidant, phytochemicals, nutritional and minerals in the fruit of Kigelia africana (Lam.) Benth
Authors: OSENI OLATUNDE ABASS 
Published: June 2021

The health problems in the third world and the attendant complications resulting into death of many people mostly caused by cancer, hypertension and other heart disorders primarily because of the inability to afford adequate health conditions and maintain balanced nutrition status has called for alternative means of attaining the required global Millennium Developmental Goals (MDGs) status on health and also to prevent mortality among all ages. This study therefore aimed at investigating the natural antioxidants, phytochemicals, proximate and mineral elements composition in fruit of Kigelia africana popularly known as the sausage tree as an indication for utilization in pharmaceutical for human and animals' feed benefits. The results obtained for 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), total phenolic content, total flavonoid, Ferric reducing antioxidant power (FRAP), phytochemical constituents, proximate and mineral elements compositions of the fruits revealed high percentage of antioxidant potentials, presence of medicinal important phytochemicals, good quantity of nutritional and medicinally important mineral elements. However, from the results of the study, the fruit of Kigelia africana can be exploited for medicine to heal many diseases and food additives in animals' feed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.