bluuebunny/crossref_metadata_2025 huggingface dataset:
A Molecular Dynamics Study of Interaction between Kinesin Light Chain and Amyloid Precursor Protein
Authors: Taylor Cooper , Shah Soleil , Miller Bill , Parish Carol
Published: June 2021

The misregulation of the cargo binding domain of kinesin‐1 light chain (KLC) has been linked to various neurodegenerative diseases, most notably Alzheimer's, as KLC may play a role in amyloid precursor protein (APP) transport and subsequent degradation. Recent literature suggests a third protein, C‐Jun‐amino‐terminal kinase‐interacting protein 1 (JIP1), acts as a mediator protein for binding of KLC to APP. Computational molecular dynamics (MD) was conducted on this ternary complex, as well as the JIP1:KLC complex, using the Amber MD package to understand the atomistic interactions between the structures. Schrodinger's Glide was used to dock the structure of JIP1 (extracted from PDB 2H96) to KLC to form the binary complex, while the online Patchdock server was used to dock APP to the JIP1:KLC complex to form the ternary state. Within simulations of various JIP1:KLC complexes, the JIP1 protein would orient itself in nearly the same location relative to KLC regardless of the initial starting conformation used for MD. The simulations were also used to help elucidate a low energy conformation of the bound complex, since this conformation has not been resolved experimentally. Preliminary binding free energy calculations suggest that the ternary complex is more stable than the binary complex (APP:KLC).
Identification of Oma1p Protease Sensitive Sites in Subunit 1 of Yeast Cytochrome Oxidase
Authors: McStay Gavin , Zafar Mujtaba , Tamanna Farhana , Thomas Brent , Ivezic Suzana , Chowdhury Ashfiyah , Noreen Saba
Published: June 2021

Cytochrome c oxidase is the terminal electron acceptor in the electron transport chain in many aerobic organisms. This multi‐protein complex can be composed of a varying number of subunits depending on species. In the budding yeast, Saccharomyces cerevisiae, cytochrome c oxidase is made up of 11 subunits, 8 are derived from the nuclear genome and 3 are derived from the mitochondrial genome. To ensure these subunits come together at the correct time and place a family of assembly factors is required. In the case of the mitochondrial DNA encoded subunit 1 there is a linear pathway of biosynthesis where these assembly factors and other structural subunits associate at specific times to regulate specific maturation events. In the absence of some assembly factors subunit 1 is subject to degradation by the protease Oma1p. In yeast, this results in a deficiency in cytochrome c oxidase activity and the inability to grow using substrates requiring intact oxidative phosphorylation. We have performed a mutagenic screen of mitochondrial DNA to identify mutations in the subunit 1 gene that can suppress the oxidative phosphorylation deficient phenotype caused by the absence of the assembly factors, COA2 and SHY1. Through analysis of the COX1 gene we will identify putative regions required for the proteolytic processing of subunit 1 by the Oma1p protease. The information gathered from this research will guide us in identifying putative regions required for cytochrome c oxidase subunit 1 degradation and also provide more information regarding the proteolytic requirements of the Oma1p protease.
Oligomerization of Two Membrane Proteins Critical for Bacterial Mating
Authors: Pinkhasov Omar , Berkmen Melanie
Published: June 2021

Bacterial mating allows for the transfer of DNA through a DNA translocation channel. These large, multi‐protein complexes can be found in many bacteria and are known as Type IV secretion systems (T4SSs). Our work focuses on the T4SS of the integrative and conjugative element ICEBs1 found in the Gram‐positive bacterium Bacillus subtilis. Two crucial components of the ICEBs1 T4SS are ConE and ConB. ConE is a VirB4‐like peripheral membrane ATPase, likely providing the energy necessary to pump DNA though the cell membrane. ConB is a VirB8‐like integral membrane protein that recruits ConE to the membrane. Our objective was to purify ConB and to characterize its oligomerization state and potential interactions with ConE in vitro using blue native polyacrylamide gel electrophoresis (BN‐PAGE). We found that purified full‐length His6‐ConB is a mixture of oligomers, forming tetramers up to dodecamers. Truncated His6‐ConB lacking its N‐terminal transmembrane segment is monomeric. Confirming a prior study, we found that purified His6‐ConE and MBP‐His6‐ConE form dimers and a mixture of higher order oligomers. No interactions were detected between any of our purified ConE and ConB proteins using BN‐PAGE. Our results on ConB and ConE multimerization are consistent with previous findings on homologous T4SS proteins. We propose that VirB8‐like proteins function as dodecamers and VirB4‐like proteins function as hexamers in assembled T4SSs in vivo. ConB and ConE likely must interact with the membrane and/or other components of the T4SS to achieve the fully oligomerized state.
Toward the Structural Characterization of the Gabapentin Binding site
Authors: Briot Julie , Sygusch Jurgen , D'avanzo Nazzareno , Parent Lucie
Published: June 2021

Gabapentinoids are prescription drugs used to relieve neuropathic pain, epilepsy and anxiety. One member of this family, gabapentin has been shown to bind with nanomolar affinity to the CaVα2δ subunit of pre‐synaptic voltage‐gated calcium channels. We undertook a study to characterize the structural determinants of the gabapentin binding site on the extracellular N‐terminal domain of CaVα2δ. The nucleotide sequence of a domain of 246 residues, including the putative binding site, was inserted in a pET28 vector. The protein, expressed in BL21(DE3) bacteria, was found to be mostly located in inclusion bodies and had to be solubilized in 8 M urea. Primary sequence of the purified protein was confirmed by MALDI mass spectrometry and protein refolding was promoted by removing urea through overnight dialysis. The recovery of the native‐like structure was validated using size exclusion chromatography, circular dichroism, and small‐angle X‐ray scattering. The protein was found to be stable at 4°C for 2 weeks. Interaction between the 246‐residue domain (2 µM) and gabapentin (2 µM, 20 µM and 40 µM) was examined using differential scanning fluorimetry. Preliminary data show that gabapentin does not significantly affect the stability of this domain. We have nonetheless undertaken crystallization trials with and without gabapentin to obtain a high‐resolution 3D structure of this domain. Other structural domains of CaVα2 are concurrently in the process of purification using similar approaches. Altogether our studies will ultimately shed light on the molecular mechanism underlying the effect of gabapentin in pain management.
Shotgun Proteomic Analysis of Protein Expression in Mosquito Ovaries Post Blood Meal
Authors: Geiser Dawn , Li Wenzhou , Pham Daphne , Wysocki Vicki , Winzerling Joy
Published: June 2021

Female mosquitoes require a blood meal for oogenesis and receive a substantial iron load in this meal in the form of holo‐transferrin and hemoglobin. During the course of digestion and absorption in the 72 hours post blood feeding, the iron concentration in the gut decreases 10‐fold while that in the ovaries more than doubles. Eggs are laid with ~125 ng Fe each; the majority coming from the blood meal. Iron is mitogenic and iron deposition in the ovaries may induce changes in the endogenous protein expression profile over time. Proteomic analysis of extracts from developing ovaries and eggs revealed proteins that are expressed at time intervals of 24 and 72 hours post blood feeding. These analyses may provide further insight into the effect of a blood meal on mosquito iron metabolism and mosquito fecundity.
Interactome Analysis of BetaM Protein Encoded by ATP1B4 Gene Using Two‐hybrid Systems
Authors: Pestov Nikolay , Korneenko Tatyana , Modyanov Nikolai
Published: June 2021

The BetaM, a type II integral membrane protein, became a muscle‐specific protein of inner nuclear membrane in eutherians through co‐option of the ATP1B4 gene that encodes Na,K‐ATPase β‐subunit in lower vertebrates. Earlier we determined that BetaM directly interacts with transcriptional co‐regulator SKIP and functions as a regulator of gene expression. Here we present the expanded search for BetaM interactors by screening of cDNA libraries using conventional yeast two‐hybrid and split‐ubiquitin systems. The list of identified BetaM interactors includes BetaM itself, SKIP, Syne‐1, sarcoglycan β, LZIP, RCN 3, LAP1, HO 1, HO2, PHD finger 3, cadherin16, frizzled 1, SC65, tetraspanins 3 and CD63, TGN29, ERGIC 3, BCRAP‐31, C4orf3 and SftpC. Only sarcoglycan β was identified by both assays. Analysis of BetaM mutants revealed that deletion of 56 N‐terminal residues including the Arg‐rich peptide and a half of Glu‐rich stretch does not disrupt the BetaM dimerization and other identified interactions. Transmembrane and/or C‐terminal domains are required for interactions with LZIP, Syne, reticulocalbin 3 and sarcoglycan β. N‐terminal domain seems to be sufficient for interaction with SKIP, LAP‐1, PHF‐3 and heme oxygenases. Further analysis indicates that the second half of the Glu‐rich intrinsically disordered N‐terminal domain is required for the interaction with SKIP. We also show that the relatively conserved fragments of N‐terminal domain adjacent to the membrane are sufficient for BetaM dimerization.

Supported by RFBR (Grant 13‐04‐01413) and by Dept of Physiology & Pharmacology, University of Toledo College of Medicine.
Using ProMol to Study Zinc Finger Motifs
Authors: Albalwi Majied , Bernstein Herbert , Craig Paul
Published: June 2021

Currently, template‐based alignments focus on detection of motifs in novel structures that correspond to specific functions. ProMOL is a plugin for the PyMOL molecular graphics environment that has been used to detect motifs for enzyme active sites. The purpose of this study is to create non‐active site structural motifs in proteins, with a focus on the Zinc finger (ZnF) families from Pfam ‐ Cys2His2, Gag knuckle, Treble clef, Zinc ribbon, and Zn2/Cys6. While these motifs are not catalytic, zinc domains contribute to structure and function of proteins involved in many essential cellular processes. The methodology begins with creation and testing of the ZnF motifs against themselves followed by testing on homologs. A series of user‐created motif templates (the U set) will be prepared and characterized. Those with high specificity and selectivity will then be further refined and form the basis for the ProMOL S set (structural motifs). These motif templates will then be used to propose functions for unannotated structures in the Protein Data Bank. This work has been funded in part by National Institute of General Medical Sciences 2R15GM078077‐02, 3R15GM078077‐02S1 and 3R15GM078077‐02S2.
Proteomic Analysis of Sera and Saliva from Children with Autism Spectrum Disorder (ASD) and Matched Controls
Authors: Wormwood Kelly , Ngounou Wetie Armand , Sokolowska Izabela , Beglinger Katherine , Ryan Jeanne , Woods Alisa , Darie Costel
Published: June 2021

Approximately 1/88 children are believed to be affected by Autism Spectrum Disorder (ASD). A more recent study suggests numbers as high as 1/50. With the increased diagnosis of ASD, treatment and understanding of the disorder is a pressing health concern. Protein biomarkers found for ASD may be used for ASD diasgnosis, subtyping, monitoring, treatment, identifying therapeutic targets and may also provide clues about the causes of ASD. Here, sera and saliva samples from children with ASD and matched controls were analyzed using a combination of gel electrophoresis, in gel digestion or in solution digestion and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate differences between the proteomes of ASD patients and matched controls. These results will hopefully shed light on possible causes of ASD. In the SDS‐PAGE and Tricine‐PAGE based experiments using sera, increased levels of Apolipoproteins (Apos) ApoA1 and ApoA4, involved in cholesterol metabolism, and of serum paraoxanase/arylsterase 1, involved in preventing oxidative damage, were discovered in the sera of children with ASD, compared with their matched controls. These three proteins are parts of High Density Lipoproteins (HDLs) and are presumed to interact with one another. This data suggests that there may be a dysregulation of cholesterol metabolism in children with ASD. Additional dysregulated proteins were also detected, suggesting a dysregulated immunological response and an enhanced response to environmental toxic factors. This investigation is ongoing.
Efficient and Convenient Enrichment of Multi‐spanning Membrane Proteins for Proteomic Studies
Authors: Geddes Joanna , Meier Scott , Cirbaite Evelina , Siurkus Juozas , Kaboord Barbara , Opperman Kay
Published: June 2021

Examining the membrane proteome is vital to understand their role in normal and disease function. However, the isolation and extraction of multi‐spanning membrane proteins for proteomic study often proves difficult. Traditional isolation methods are tedious and time‐consuming. Additional drawbacks include poor solubilization, incompatibility with downstream applications, and disruption of membrane protein complexes. We report a procedure that enables fast, convenient solubilization of membrane proteins from cultured cells and tissue. This sequential detergent extraction method allows for the enrichment of both integral membrane proteins and membrane‐associated proteins. To validate our method, three integral multi‐spanning membrane proteins ADP/ATP translocase 3 (SLC25A6), sodium/potassium ATPase alpha subunit (AT1A1) and adenylate cyclase 2 (ADCY2)were evaluated. Membrane protein extraction in cell lines (A431, HeLa, HCT116, HepG2, HEK293 and A549) and mouse tissue (brain, kidney and liver) using this reagent‐based extraction outperforms the other available kits as determined by western blotting and mass spectrometry. Additionally, extraction of multi‐spanning membrane protein is improved by increasing the ionic strength of the solubilization buffer, obtaining 70‐85% extraction, as confirmed by western blot densitometry. Native membrane protein complexes are also preserved, allowing for compatibility with co‐immunoprecipitation experiments. This straight forward and robust method enables researchers to better investigate the role of membrane proteins in cellular functions.
Investigation of Tumor Differentiation Factor (TDF)‐induced Cell Differentiation using Mass Spectrometry
Authors: Channaveerappa Devika , Ngounou Wetie Armand , Lupu Loredana , Robu Adrian , Zamfir Alina , Darie Costel
Published: June 2021

Tumor differentiation factor is a protein, produced by the pituitary gland and is secreted into the bloodstream. TDF is known to induce differentiation in human breast and prostate cancer cells and not in other cells. However, the site of action or receptor and the mechanism of action of TDF to induce differentiation are unknown. In recent studies done by our group, potential TDF receptor candidates in human breast and prostate cancer cells were identified. Substantial differences were also observed in the SDS‐PAGE protein patterns of untreated and cancer cells treated with TDF analogs. Hence, the current study is focused on the proteomic analysis of TDF‐untreated and TDF treated MCF7 and DU145 human breast and prostate cancer cells, on functional investigation of TDF as well as on the localization of TDF. These experiments will help elucidate the mechanism through which TDF induces cell differentiation. To carry out these investigations, MCF‐7 human breast and DU‐145 human prostate cancer cells were grown under standard cell culture conditions. The cells were treated with various concentrations of TDF analogs(0 to 10 µg), lysed and the proteins were separated by SDS‐PAGE and stained with Coomassie for qualitative inspection and direct comparison of the samples. The gel bands were cut into pieces which were then digested and the different peptide mixtures were analyzed by nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) using a nanoAcquity UPLC coupled with a QTOF Micro mass spectrometer, followed by data analysis using MassLynx (version 4.1), ProteinLynx Global Served (PLGS 2.4), Mascot and Scaffold 3.0. Further validation and functional studies will also be performed.
Effects of Anesthetics Pentobarbital Sodium and Chloral Hydrate on Urine Proteome
Authors: Zhao Mindi , Gao Youhe
Published: June 2021

Urine can be a better source than blood for biomarker discovery since it accumulates many changes. The urine proteome is susceptible to many factors such as anesthesia. Pentobarbital sodium and chloral hydrate are commonly used anesthetics in animal experiments. The relative abundance of 22 and 23 urinary proteins were changed with pentobarbital sodium or chloral hydrate anesthesia, respectively, as determined by label‐free quantification. Seven proteins had the same trends with two anesthetics. Among these changed proteins, some had been considered as candidate biomarkers such as uromodulin, some had been considered stable in healthy human urine, which are more likely to become biomarkers when changed, such as transferrin. The pattern of showed urinary proteins provides clues to the discovery of urinary proteins regulatory mechanisms. Anesthetic‐related effects can be excluded in future biomarker discovery studies.
Whole Exome Sequencing Analysis Is an Efficient Method for Comprehensively Evaluating Genetic Variability Associated with Interparticipant Response to Glucocorticoid Receptor‐Mediated Signaling
Authors: Lassalle‐Klein Rose , Forst Thomas , Knox Rachel , Murphy Patrick
Published: June 2021

The goal of this study was to identify correlations between interpersonal genetic variability and alterations in the intracellular glucocorticoid receptor (GR) signaling response to mild cardiovascular stress. Circulating cortisol concentrations in healthy volunteers varied significantly among participants, both before and after HPA axis stimulation. These differences could not be correlated with biobehavioral survey responses or targeted Sanger sequencing of genes encoding the GR, hsp90, or CRHR1. We consequently hypothesized that whole exome sequencing (WES) would provide a feasible approach for determining variability among phenotypic groups and revealing causal gene candidates that had not been previously considered. WES was conducted using genomic DNA from four participants, one showing the canonical high cortisol response and three showing an enigmatic negative response. Using an Agilent V4 exome enrichment platform and Illumina HiSeq2000 paired end sequencing, >44 million reads per sample were aligned to the hg19 reference build. Comparative analysis of opposing phenotypes showed a variance profile of mutations associated with the negative cortisol response that warrant further investigation. We predict that the statistically significant genetic variants shared among negative cortisol responders may be associated with critical changes in the GR‐mediated signaling pathway and alter a patient's response to stress and glucocorticoid‐based pharmacotherapies.
Peptidomic and Proteomic Profiling of the Juvenile Rheumatoid Arthritis (JRA) Synovial Fluid Reveals Transthyretin (TTR) as Potential Biomarker of the Disease
Authors: Clement Cristina , Moncrieffe Halima , Becerra Aniuska , Stern Lawrence , Santambrogio Laura
Published: June 2021

Juvenile rheumatic arthritis is the most common chronic condition seen by pediatric rheumatologists. There are high concentrations of immune complexes in the joint but their specificities are just recently being investigated. The presented research was undertaken to compare the amount of autoantigens and autoantibodies in the synovial fluid (SF) of JRA patients in comparison to healthy controls. SF samples were collected from 37 JRA and 10 healthy volunteers. The SF peptidome was fractionated by ultrafiltration and peptides with MW less than 10 kDa were sequenced by HCD/ETD nanoLC Orbitrap‐ESI‐MS/MS. Protein A&G gels were used to purify the immune complexes from SF followed by acidic elution of the IgG bound‐proteins. The eluted and the total SF proteomes were fractionated by one‐dimensional gel electrophoresis (1DEF) nanoLC Orbitrap‐ESI‐MS/MS. Ingenuity pathway analysis highlighted the biochemical pathways of JRA SF: acute phase response signaling, complement activation, coagulation system among others. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed up‐regulated expression in the peptidome and the Ig‐G bound proteome from the SF of JRA patients. The production of autoantibodies was validated by ELISA and Western blot analysis using recombinant protein TTR. These data support the TTR as a potential biomarker for the pathogenesis, diagnosis and progression of JRA disease.
Prediction of a new SelD‐like family that may participate in sulfur metabolism in hyperthermophilic sulfur‐reducing archaea
Authors: Zhang Yan
Published: June 2021

Selenium (Se) and sulfur (S) are closely related elements that exhibit similar chemical properties. Some genes related to S metabolism are also involved in Se utilization in many organisms. However, the evolutionary relationship between the two utilization traits is unclear. In this study, we conducted a comparative analysis of the selenophosphate synthetase (SelD) family, a key protein for all known Se utilization traits, in all sequenced archaea. Our search showed a very limited distribution of SelD and Se utilization in this kingdom. Interestingly, a SelD‐like protein was detected in two orders of Crenarchaeota: Sulfolobales and Thermoproteales. Genomic and phylogenetic analyses revealed that SelD‐like protein may share a common ancestor with SelD and might be involved in S metabolism in these S‐reducing organisms. Further genome‐wide analysis of patterns of genes occurrence in different thermoproteales suggested that several genes, including SirA‐like, Prx‐like and adenylylsulfate reductase, were strongly related to the SelD‐like protein. Finally, we proposed a simple model wherein the SelD‐like protein may play an important role in the biosynthesis of certain thiophosphate compound. Our data suggest novel genes involved in S metabolism in hyperthermophilic S‐reducing archaea, and may provide a new window for understanding the complex relationship between Se and S metabolism in archaea.
The Effect of Divalent Cations on Complex Phosphoinositide/Lipid Monolayers at the Air/Water Interface
Authors: King Katrice , Qifti Androniqi , Gericke Arne
Published: June 2021

Objective

Investigate the effect of common inner leaflet plasma membrane lipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), on phosphoinositide domain behavior in the presence of cholesterol and/or divalent cations.

Background

PI and phosphoinositides (PIPs) may only comprise a small percentage of the inner leaflet of the plasma membrane, but they have major implications in a variety of signaling events. Previously, we have observed the absence of macroscopically discernible domains in mixtures of PI/PE and PI(4,5)P2/PE. Upon the addition of cholesterol to these mixtures, we observe condensation of the monolayer and domain formation.

Methods

We use epifluorescence microscopy to image domain formation in lipid monolayers at the air/water interface

Results

We find that the addition of varying concentrations of Ca2+ to PE/PI(4,5)P2/Cholesterol monolayers result in a decrease in the size of the domains. Our hypothesis is that this is due to a penetration of the Ca2+ ions into the PI(4,5)P2 headgroup region, leading to a disruption of the hydrogen bond network formed by the PI(4,5)P2 headgroup and cholesterol. For both PE/PI(4,5)P2 and PI/PI(4,5)P2, we find homogeneous mixing of the monolayer. Alternatively, when cholesterol is added to PE/PI(4,5)P2 we observe the formation of small domains at low pressures. In addition to cholesterol, we also investigate the effect of divalent cations on these lipid mixtures.
Regulation of Yeast Glycerol‐3‐Phosphate Acyltransferase Gpt2p/Gat1p Through Phosphorylation
Authors: Shabits Brittney , Nagler Bernadette , Athenstaedt Karin , Zaremberg Vanina
Published: June 2021

Glycerol‐3‐phosphate acyltransferases (GPAT) catalyze the acylation of glycerol‐3‐phosphate, the first and rate limiting reaction in de novo synthesis of phosphatidic acid (PA). GPAT redundancy led to the hypothesis that isoenzymes are forming different PA pools which supply different pathways. In the model organism S. cerevisiae two GPATs have been identified, Gpt2 and Sct1. Most recently we demonstrated the important role of Gpt2 (but not Sct1) in cells challenged by oleic acid as the only carbon source. Whereas growth in oleic acid normally induces lipid particle accumulation, cells lacking GPT2 failed to accumulate these neutral lipids storage compartments, exhibited a strong growth defect, and cell viability was reduced. The fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under this growth condition attracted our interest. We have identified 3 highly conserved phosphorylation sites in Gpt2 by mass spectrometry, localized to a Ser rich region in the carboxy‐end. Ser to Ala triple mutations as well as deletion of the Ser rich region, were introduced in the yeast genome to investigate the role of phosphorylation in the regulation of Gpt2. Importantly, both Gpt2 mutant forms retain enzymatic activity sufficient to support life of cells lacking Sct1. Microscopic inspection of phosphomutants revealed changes in lipid droplet formation. In line, cells overproducing these mutants exacerbated this phenotype, suggesting that lack of Gpt2 phosphorylation leads to abnormal accumulation of lipid droplets and carbon source consumption. The role of the Gpt2 serine‐rich carboxy‐end in mediating protein‐protein interactions is currently investigated
Yeast Nem1‐Spo7 Protein Phosphatase Activity on Pah1 Phosphatidate Phosphatase is Specific for the Pho85‐Pho80 Protein Kinase Phosphorylation Sites
Authors: Su Wen‐Min , Han Gil‐Soo , Carman George
Published: June 2021

Pah1 is the phosphatidate phosphatase in the yeast Saccharomyces cerevisiae that produces diacylglycerol for triacylglycerol synthesis and concurrently controls the levels of phosphatidate used for phospholipid synthesis. Phosphorylation and dephosphorylation of Pah1 regulate its subcellular location and phosphatidate phosphatase activity. Compared with its phosphorylation by multiple protein kinases, Pah1 is dephosphorylated by a protein phosphatase complex consisting of Nem1 (catalytic subunit) and Spo7 (regulatory subunit). In this work, we characterized the Nem1‐Spo7 phosphatase complex for its enzymological, kinetic, and regulatory properties with phosphorylated Pah1. The dephosphorylation of Pah1 by Nem1‐Spo7 phosphatase resulted in the stimulation (6‐fold) of phosphatidate phosphatase activity. For Pah1 phosphorylated by the Pho85‐Pho80 kinase complex, maximum Nem1‐Spo7 phosphatase activity required Mg2+ ions (8 mM) and Triton X‐100 (0.25 mM) at pH 5.0. The energy of activation for the reaction was 8.4 kcal/mol, and the enzyme was thermally labile at temperatures above 40 °C. The enzyme activity was inhibited by sodium vanadate, sodium fluoride, N‐ethylmaleimide, and phenylglyoxal, but was not significantly affected by lipids or nucleotides. Nem1‐Spo7 phosphatase activity was dependent on the concentrations of Pah1 phosphorylated by Pho85‐Pho80, Cdc28‐cyclin B, PKA, and PKC with kcat and Km values of 0.29 s‐1 and 81 nM, 0.11 s‐1 and 127 nM, 0.10 s‐1 and 46 nM, and 0.02 s‐1 and 38 nM, respectively. Its specificity constant (kcat/Km) for Pah1 phosphorylated by Pho85‐Pho80 was 1.6‐, 4‐, and 6‐fold higher, respectively, than that phosphorylated by PKA, Cdc28‐cyclin B, and PKC.
Signaling Regulates Activity of DHCR24, the Final Enzyme in Cholesterol Synthesis
Authors: Luu Winnie , Zerenturk Eser , Kristiana Ika , Sharpe Laura , Brown Andrew
Published: June 2021

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis (3β‐hydroxysterol Δ24‐reductase; DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its post‐translational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell‐line (Chinese hamster ovary‐7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling. Moreover, we propose that post‐translational modifications such as phosphorylation are a valuable resource for mapping the topology of membrane‐associated proteins such as DHCR24.

The Brown lab is funded by grants from the National Health and Medical Research Council.
Inhibition of Apoptosis by Angiotensin 1‐7/mas Pathway through MKP‐2 Activation in Alveolar Epithelial Cells
Authors: Gopallawa Indiwari , Uhal Bruce
Published: June 2021

Study Objective:

The autocrine generation of angiotensin II (ANGII) and c‐Jun‐N‐ terminal kinase phosphorylation (pJNK) are both required in alveolar epithelial cell (AEC) apoptosis which contributes to lung diseases. Angiotensin 1‐7 (ANG 1‐7) protects against AEC apoptosis which is induced by ANG II. However the exact inhibitory mechanisms of ANG 1‐7 and the mas receptor are currently unclear in AECs. The objective of the current research is to identify the role of map kinase phosphatase (MKP‐2) in AEC survival. We hypothesized that ANG 1‐7 activates MKP‐2 and maintains low pJNK levels thereby preventing apoptosis.

Methods

To determine the functional role(s) of MKP‐2, AECs were transfected with MKP‐2 siRNAs in the presence of ANG II (0.1µM) and/or ANG I‐7 (0.1µM). Then the cells were subjected to determine pJNK and apoptosis by Western blotting and nuclear fragmentation assay respectively. The loss of mitochondrial membrane potential (MMP) was also measured using a lipophilic probe. To test the involvement of mas receptor the cells were transfected with anti‐sense oligonucleotides in the presence of ANG 1‐7 followed by Western blotting to detect MKP‐2

Results

The current data show the induction of pJNK (p<0.01), nuclear fragmentation (p<0.01) and MMP (p<0.001) when MKP‐2 is knocked down. The data also show that silencing MKP‐2 prevented the blockade of apoptosis by ANG 1‐7. Moreover the blockade of the mas receptor by the antagonist (A779) or anti‐sense oligonucleotides attenuated the induction of MKP‐2 by ANG 1‐7

Conclusions

The results from these studies suggest that ANG 1‐7/mas activation prevents JNK phosphorylation by activating the JNK selective phosphatase MKP‐2 and further it shows the involvement of mas receptor in MKP‐2 activation.
Flow Cytometry Cell Cycle Analysis of Apoptotic Mouse HT‐2 T Lymphocytes
Authors: Pinkston Mary , Lauzon Robert
Published: June 2021

Apoptosis is an essential form of programmed cell death (PCD) in multicellular animals, in which cells are dismantled and disposed of in a controlled fashion. In the vertebrate immune system, apoptosis prevents the emergence of autoreactive lymphocytes, and is essential for self‐tolerance. In mouse HT‐2 T lymphocytes, apoptosis and internucleosomal DNA fragmentation can be initiated by cytokine deprivation (interleukin‐2 or IL‐2), addition of the protein kinase inhibitor staurosporine, or addition of the topoisomerase inhibitor camptothecin. We used flow cytometry to determine if apoptosis in HT‐2 cells involved accumulation in a specific phase of the cell cycle, and whether the mode of induction affected cell cycle distribution. Our findings indicate that IL‐2 deprivation induces cell cycle arrest in the G1 phase, prior to the accumulation of cells with sub‐G1 DNA content. In contrast, HT‐2 cells treated with staurosporine and camptothecin accumulated in G2/M and S phase, respectively. We used western blotting and quantitative chemiluminescence to demonstrate that Kip‐1 (p‐27), a CDK‐inhibitor that regulates the progression of cells from G1 to S phase, is up‐regulated in a time‐dependent fashion following IL‐2 deprivation and staurosporine. When compared to HT‐2 control cultures (+IL‐2) and Beta‐tubulin protein levels, Kip‐1 levels were significantly increased 24 hours following IL‐2 deprivation, and 6 hours post‐staurosporine addition. These findings suggest that Kip‐1 up‐regulation may be involved in multiple apoptotic pathways of T lymphocytes. Studies are currently underway to quantitatively determine protein levels of S (PCNA) and M (phospho‐histone H3) phase‐specific markers in HT‐2 cells in response to apoptotic stimuli.
Arginyltransferase (Ate1) is a Pro‐apoptotic Enzyme Essential for Stress Response
Authors: Kumar Akhilesh , Birnbaum Michael , Zhang Fangliang
Published: June 2021

Arginylation consists of addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence as well as the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. By using multiple types of eukaryotic cells as test models, we found that Ate1 is a pro‐apoptotic factor. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of various stressing factors including heat shock, oxidative and osmotic stress, and heavy metal. Oppositely, over‐expression of Ate1 leads to cell death in an Aif1‐dependent and caspase‐independent manner. Our data further suggest that localization of Ate1 to mitochondria and its enzymatic activity is essential for apoptosis during stress. Intriguingly, we also found that the activity of arginylation is essential for the wellbeing of the cells during mitochondria‐dependent metabolism as well as nutrient‐deprivation. Our data suggests that arginylation is a global regulator for metabolism and stress response, and provides an explanation for the observed role of Ate1 in carcinogenesis and injury.
Investigating the Role of the Yeast Metacaspase, Yca1p, in the Programmed Cell Death of Aneuploid Cells of the Budding Yeast, Saccahromyces cerevisiae
Authors: Rogers Stephen , Frazier Ryan , Pinches Seth , Austriaco Nicanor
Published: June 2021

Aneuploidy is the genetic state of a cell that has a chromosomal number that is not an exact multiple of the haploid complement. It is a leading cause of spontaneous abortions and of mental retardation in humans, and is a characteristic defect in cancer. Yeast cells that are aneuploidy manifest a diversity of phenotypes including cell cycle defects, genomic instability, protein imbalance, chaperone stress and proteotoxicity. We are investigating the links between aneuploidy on the yeast S. cerevisiae and programmed cell death. We have generated aneuploid yeast cells that either lack the yeast metacaspase, Yca1p, or contain a GFP‐tagged Yca1p, and are characterizing the behavior of these cells in response to apoptotic‐stimuli. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]
Posttranslational Modification Cross Talk Regulates the Critical Adaptive Immune Molecule, The Class II Transactivator
Authors: Morgan Julie , Greer Susanna
Published: June 2021

Major histocompatibility class II (MHC II) molecules present pathogenic and tumorigenic antigens to CD4+T cells and play vital roles in regulating adaptive immune responses. MHC II is controlled at the level of transcription by the Class II Transactivator (CIITA), which in turn is regulated through a series of posttranslational modifications (PTMs). Acetylation of CIITA by the histone acetyltransferase (HAT) pCAF, results in CIITA nuclear localization. CIITA is also phosphorylated by Extracellular Signal Regulated Kinase (ERK1/2) at S280, a critical site for regulating additional PTMs such as mono‐ubiquitination. Both phosphorylation and mono‐ubiquitination is key in CIITA's stability and increased transactivation. We have identified that phosphorylation of CIITA by ERK1/2 enhances K63 linked ubiquitination leading to CIITA's ability to bind to the MHC II promoter. pCAF was recently identified as an E3 ligase for the nuclear protein Mdm2. We show here pCAF increases both CIITA transactivation and ubiquitination; suggesting pCAF also plays a role in regulating CIITA ubiquitination whether through its HAT or E3 ligase capability has yet to be elucidated. These data provide novel links between acetylation, phosphorylation and ubiquitination of this critical inflammatory protein. Our data suggests particular posttranslational modifications of CIITA result in very different functional outcomes, and may enable the identification of biomarkers for immune diseases associated with dysregulated antigen presentation. Support for research provided by Molecular Basis of Disease area of Focus, Georgia State University
Proteomic Mapping of Mitotic O‐GlcNAc Sites
Authors: Whelan Stephen , Tan Ee Phie , Lanza Chris , Costello Catherine , McComb Mark , Slawson Chad
Published: June 2021

Regulated mitotic progression coupled with high‐fidelity chromosome segregation is crucial for normal cellular division. Phosphorylation. Although, phosphorylation plays a critical role regulating mitosis, it alone cannot account for all the complexities of mitosis. We have demonstrated that O‐GlcNAcylation is critical for proper mitotic regulation and disruptions in O‐GlcNAc signaling lead to aberrant cell division. O‐GlcNAc (b‐N‐acetylglucosamine) is an ubiquitous protein modification consisting of a single N‐acetylglucosamine residue attached to Ser or Thr in cytoplasmic and nuclear proteins in response to changes in the cellular environment. Alterations to the proper rate of O‐GlcNAc cycling lead to mitotic defects. The need for detailed O‐GlcNAc site‐mapping information is paramount for our understanding of the role of O‐GlcNAc in regulating mitotic progression. We have developed a combination of tools and methods to site map O‐GlcNAc using a Q Exactive hybrid Quadrupole‐Orbitrap Mass Spectrometer. Interestingly, we have found the Repo‐Man protein to be modified by O‐GlcNAc. Repo‐man is a Protein Phosphatase 1γ binding protein that recruits the phosphatase to the spindle in order to antagonize the function of the spindle kinase Aurora B (AurB). Repo‐man is critical for proper spindle development; loss of Repo‐man expression is lethal. Together, our data demonstrate an efficient and robust method to map O‐GlcNAc sites. The identification of O‐GlcNAc modified mitotic proteins will provide new context as to how O‐GlcNAc cycling regulates mitotic progression and spindle development. NIH‐NHLBI Contract No. HHSN268201000031C, NIH‐GM grant P41 GM104603 and DK100595
O‐GlcNAcylation Regulates Ischemia‐Induced Neuronal Apoptosis Through AKT Signaling
Authors: GONG Cheng‐Xin , Shi Jianhua , Gu Jin‐hua , Dai Chunling , Gu Jianlan , Jin Xiaoxia , Sun Jianming , Iqbal Khalid , Liu Fei
Published: June 2021

Apoptosis plays an important role in ischemia‐induced neuronal death and the loss of neural function of the affected area. The molecular mechanism by which cerebral ischemia causes apoptosis remains elusive. In this study, we found that O‐GlcNAcylation, a unique protein posttranslational modification with O‐linked β‐N‐acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co‐immunoprecipitation and mutagenesis techniques, we identified O‐GlcNAc modification likely at both Thr308 and Ser473 of AKT. O‐GlcNAcylation‐induced apoptosis was attenuated by over‐expression of AKT in cultured cells. We further found a dynamic elevation of protein O‐GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O‐GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O‐GlcNAcylation in ischemic mouse brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O‐GlcNAcylation that promotes apoptosis through down‐regulation of AKT activity. These findings provide a novel mechanism through which O‐GlcNAcylation regulates ischemia‐induced neuronal apoptosis through AKT signaling.
Using Thiol‐Reactive Cross‐Linkers to Stabilize Interactions between Thioredoxin and Its Redox Partners in Baker's Yeast
Authors: Allan Kristin , Pilat Jennifer , Chepngeno Juliet , Sizek Herbert , West James
Published: June 2021

Increased levels of reactive oxygen species can lead to a variety of physiological and pathophysiological effects, largely resulting from damage to biological macromolecules. To prevent oxidative damage on cysteine residues in proteins, cells have evolved a specific redox network that uses the enzyme thioredoxin to reduce disulfide bonds. In earlier experiments, we found that the baker's yeast thioredoxin Trx2 undergoes cross‐linking with two of its redox partners (its major substrate (the peroxiredoxin Tsa1) and thioredoxin reductase (Trr1)) in cells treated with bifunctional electrophiles. In subsequent experiments, we found that this cross‐linking was due to modification of the active cysteine residues in these proteins. Here, we have expanded our study to determine if thiol‐reactive cross‐linkers can be used to stabilize interactions between Trx2 and a broader number of its substrates. We have found that several thioredoxin substrates including the peroxiredoxins Tsa1 and Tsa2, the methionine sulfoxide reductase Ykg9, and the sulfiredoxin Srx1 immunoprecipitate in cross‐linked complexes with Trx2 following treatment of cells with the cross‐linker divinyl sulfone (DVSF). Moreover, several of the cross‐linked species that form in these substrate proteins are absent in yeast deficient in cytosolic thioredoxins, implying specific cross‐linking with thioredoxin. These results indicate that protein cross‐linkers stabilize interactions between thioredoxin and its redox partners, a finding that may be useful in identifying novel interaction partners in other cellular redox networks.
Writing and Reading the CTD Code: In Vitro Reconstruction of Post‐Translational Modifications on RNA Polymerase II
Authors: Mayfield Joshua , Robinson Michelle , Brodbelt Jennifer , Zhang Yan
Published: June 2021

Information flow within the cell is filtered through a vast array of codes. Vital biological data is compiled at the DNA, RNA, and protein levels with additional information relayed through an ever‐expanding repertoire of post‐transcriptional and post‐translational modifications (PTM). The PTM state of proteins can impact their recognition, structure, and function as well as relay important signals to influence cellular events, as found in histone code. A newly characterized coding system in eukaryotic transcription is “CTD code.” The C‐terminal Domain of RNA Polymerase II (CTD) is a unique structure composed of repeats of the consensus heptad YSPTSPS. These residues are subject to a variety of PTMs and specific factors are recruited depending on the PTM state of CTD. Particular PTM marks are linked to transcriptional events including initiation, elongation, and termination. The PTM state of CTD is generated and altered through the action of PTM enzymes such as kinases and phosphatases. Current technologies allow the determination of isolated and simplistic PTM marks along the CTD but lack the potential for the investigation of a combinatorial code or the identification of novel PTM marks. To overcome this we propose a bottom up approach to in vitro reconstruct the CTD code. We treat CTD substrate with PTM enzymes and characterize resultant modifications using a combination of molecular biology and mass spectrometry. Our novel mass spectrometry methodologies allow for heptad level resolution and characterization of individual and combinatorial PTM marks along CTD. Using this interdisciplinary cipher we can determine the lexicon for CTD code and better understand the impact of PTM on cellular communication.
Validation of Recombinant Antibodies Against Human Transcription Factors
Authors: Griffin Carly , Miersch Shane , Marcon Edyta , Banerjee Sunandan , Wells Jim , Hornsby Michael , Kossiakoff Anthony , Koide Shohei , Paduch Marcin , Sidhu Sachdev , Moffat Jason
Published: June 2021

The Recombinant Antibody Network (RAN) is a pilot project involving 3 integrated automation centres to create renewable, open‐source, high‐quality binding reagents. The main aims of RAN are to: generate rAbs (recombinant antibodies) directed to the ~1500 human transcription factors (TFs) against ~3000 domain targets using high‐throughput selection methods; validate and produce 蠅2 rAbs per target for high performance in applications such as immunofluorescence (IF), western blot, and chromatin‐immunoprecipitation; distribute validation data to our network and the scientific community through a dedicated web portal; and continually improve the efficiency, speed, cost and success. The RAN pipeline progressively identifies clones that bind target domains with high affinity and specificity. Our rAbs should be able to bind and IP full‐length cellular TFs from complex mixtures, and demonstrate specificity between family members. Additional validation steps include over‐expression of the target, knock‐down of the target by RNAi, and altered growth conditions. A key step in the validation process is high‐content screening against a panel of 6 Human cell lines to monitor cellular localization. The rAbs that show nuclear localization by IF are tested by immunoprecipitation‐mass spectrometry for their ability to pull‐down endogenous target. To date, over 1200 rAbs against >250 targets have been monitored by IF, with approximately 25% showing nuclear localization patterns.
Outer Membrane Protein A (OmpA) of Shigella flexneri 2a Induces B Cell Activation And Protective Immune Response in Mouse Model
Authors: Bhowmick Rajsekhar , Chakrabarti Manoj
Published: June 2021

B cells activation has a pivotal role in developing an effective vaccine as it differentiates into plasma and memory cells that conferred long lasting immunity to the host. Our earlier study established Outer membrane protein A (OmpA) of Shigella flexneri 2a as a potential vaccine candidate against shigellosis and reveals intranasal immunization of mice with OmpA of Shigella flexneri 2a elicits antigen specific IgG & IgA production, highlighting role of B cell activation in OmpA mediated immune response. The present study was undertaken to investigate whether OmpA can affect B cell activation and identify the underlying signalling pathway associated with OmpA induced B cell immune response. We have found that OmpA is recognised by TLR2 on B cell to induce secretion of protective cytokines IL6 & IL10 and promotes increased surface expression of MHCII and CD86 on B cells. After binding to TLR2, OmpA activates downstream signalling molecules like protein tyrosine kinase, phosphorylate ERK1/2 and IκB‐α thus prompting nuclear translocation of NF‐κB in B cells. Moreover we have identified OmpA induced cytokine secretion is dependent on PTKs activity ERK and NF‐kB whereas B cell differention is dependent on PTKs activity and ERK. We have also evaluated the protective efficacy of OmpA in mouse pulmonary model stating that intranasal immunization of mice with OmpA induces strong protective immunity to pulmonary pneumonia as well as robust levels of systemic and mucosal immunity against lethal challenge of four Shigella sp. Collectively, this finding strengthens the implication of OmpA as a candidate vaccine antigen against shigellosis.