bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitin chain trimming recycles the substrate binding sites of the 26 S proteasome and promotes degradation of lysine 48‐linked polyubiquitin conjugates
Authors: Liu Chang-Wei , Zhang Nan-Yan , Jacobson Andrew D , MacFadden Andrea
Published: June 2021

The 26 S proteasome possesses two distinct deubiquitinating activities: the ubiquitin (Ub) chain amputation activity removes the entire polyUb chain from the substrates; the Ub chain trimming activity progressively cleaves a polyUb chain from the distal end. The Ub chain amputation activity mediates degradation‐coupled deubiquitination. The Ub chain trimming activity can play a supportive or an inhibitory role in degradation, likely depending on features of the substrates. How Ub chain trimming assists degradation is not clear. We find that inhibition of the chain trimming activity of the 26 S proteasome with Ub aldehyde significantly inhibits degradation of Ub4 (K48)‐UbcH10, and causes accumulation of free Ub4 (generated from chain amputation) that can be retained on the proteasome. Also, a non‐trimmable K48‐mimic Ub4 efficiently targets UbcH10 to the 26 S proteasome, but it can not support efficient degradation of UbcH10 compared to regular K48 Ub4. These results indicate that polyUb chain trimming promotes proteasomal degradation of K48‐linked substrates. Mechanistically, we propose that Ub chain trimming cleaves the proteasome‐bound K48‐linked polyUb chains, which vacates the Ub binding sites of the 26 S proteasome, thus allowing continuous substrate loading.
A Comprehensive Analysis of Lysine Acetylation in Ribosomal Proteins
Authors: Koc Hasan , Cimen Huseyin , Stallard Adam , Koc Zeynep C , Koc Emine C
Published: June 2021

Protein synthesis is one of the most conserved processes in living organisms; specifically, the conservation between mammalian mitochondrial and bacterial translation machineries/ribosomes has been the most surprising one of all. Our comprehensive studies of post‐translational modifications (PTMs) to ribosomal proteins and large‐scale mass spectrometry (MS)‐based PTM mapping studies from several other laboratories suggest that the mitochondrial and bacterial ribosomes are highly modified by acetylation at their ε‐amino groups of Lys residues. The majority of the modified proteins were found to be located in the essential regions of ribosome function including, mRNA‐ and tRNA‐binding path, peptide exit tunnel, and the L1 and L7/L12 stalks. Interestingly, not only the proteins with conserved homologous functions but also the conserved residues are post‐translationally modified in both systems. The nature of this modification in both mitochondrial and bacterial ribosomes indicates that it is an essential and evolutionarily conserved mechanism for the regulation of protein synthesis.

Supported by NIH grant R01GM071034 (E.C.K.).
Characterization of npl3‐95 as a Prion‐Like Protein Involved in Translation Termination in Saccharomyces cerevisiae
Authors: Bracho Dina Paola , Correa Maria E. , Lasalde Clarivel , González Jose A. , Estrella Luis A. , González Carlos I.
Published: June 2021

Npl3 is a yeast protein that participates in various steps of mRNA biogenesis including: transcription, RNA export, processing and translation. Npl3 contains a N‐terminal region with a high glutamine and asparagine content. It also harbors a PXXP domain implicated in protein‐protein interactions. Our previous studies showed that the npl3‐95 allele within the RNA recognition motif 2 of Npl3 affects translation termination accuracy. This mutation causes the formation of high‐molecular weight (HMW) complexes not associated with polysomes. The aim of this project is to study if these novel HMW complexes are prion aggregates and their relationship with translation termination fidelity. To test this, we determined cytoplasmic inheritance of the mutant strain using cytoduction. We examined the sensitivity of the npl3‐95 strain and cytoductants to paromomycin. To evaluate the translation termination of cytoductants, the ade2‐1 analysis was also used. Yeast defective in translation termination allowed adenine synthesis and growth in media lacking adenine. Notably, the phenotypes observed in the npl3‐95 strain were “cured” by guanidine hydrochloride treatment and transferred through cytoplasmic inheritance. These results suggest that the mutant form encoded by the npl3‐95 allele acts as a prion‐like protein and further establishes a novel mechanism to regulate translation termination fidelity in yeast.
Unique biogenesis of carbon‐nitrogen‐cleaving enzyme harnessing a new mechanism
Authors: KOBAYASHI Michihiko , Zhou Zhemin , Hashimoto Yoshiteru
Published: June 2021

Nitrile hydratase (NHase) catalyzes hydration of nitrile (R‐CN) to amide. Rhodococcus rhodochrous J1 produces two NHases (H‐NHase and L‐NHase), depending on an inducer in the presence of cobalt (Co). Both enzymes have cobalt as an active center. H‐NHase is inducibly overexpressed ~50% of all soluble protein by the addition of urea to the media. It is used for the industrial production of acrylamide and nicotinamide. We discovered a novel post‐translational maturation in both NHases. The incorporation of Co into NHase depended on the α‐subunit exchange between Co‐free NHase (apo‐NHase) and its Co‐containing mediator, this unique mechanism having been named “self‐subunit swapping” and the corresponding mediator (NhhAG [consisting of NhhG and H‐NHase α ‐subunit] and NhlAE [consisting of NhlE and L‐NHaseα‐subunit]) having been recognized as a subunit swapping chaperone. We also discovered that Co was inserted into both the Co‐free apo‐NhlAE (or apo‐NhhAG) and the Co‐free apo‐α‐subunit in an NhlE (or NhhG)‐dependent manner in the presence of Co and DTT in vitro. MALDI‐TOF MS analysis revealed that the non‐oxidized cysteine residues in apo‐NhlAE and apo‐NhhAG were post‐translationally oxidized after Co insertion. These findings suggested that both NhlE and NhhG show two activities, i.e., Co insertion and cysteine oxidation. They functioned not only as a self‐subunit swapping chaperone but also a metallochaperone that includes a redox function. Both NhlAE and NhhAG consisted of heterotrimers (α e2 and α g2), and the resultant NhlE isolated from NhlAE exhibited a dimeric assembled form (e2), indicating that the structures of αe2 and αg2 would be α‐e2 (not e‐a‐e) and α‐g2 (not g‐α‐g), respectively. Current works focus now on the elucidation of the swapping mechanism.
Kinetic Analysis and Antitumor Activity of Thiosemicarbazone Benzophenone Inhibitors of cathepsin L
Authors: Chavarria Gustavo E , Strecker Tracy E , Kumar G. D Kishore , Jones Lindsay , Chaplin David J. , Pinney Kevin G , Trawick Mary Lynn
Published: June 2021

Breast cancer is the most common type of cancer found in female patients. Thus, there is an extensive search of new chemotherapeutic agents that delay or arrest cancer metastasis, the main cause of death in cancer patients. Cathepsin L overexpression has been found in numerous cancer cell lines, and in breast cancer. Effective inhibition of this cysteine protease may reduce tumor growth and metastasis. Two functionalized benzophenone thiosemicarbazones (Siles, R. et al., Bioorg. Med. Chem. Lett., 2006, 16: 4405 and Kishore, G.D. et al., Bioorg. Med. Chem. Lett., 2010, 20: 6610 ) have shown excellent in vitro inhibitory activity against human cathepsin L. Kinetic analysis and mode of inhibition of these analogs have been characterized using human cathepsin L. The ability of these inhibitors to affect the degradation of collagen by cathepsin L has been investigated. Cell invasion and migration were inhibited using MDA‐MB‐231 cells, a breast cancer cell line, that had been exposed to these synthetic compounds. (This study was supported by Oxigene, Inc.)
Molecular characterization of C. elegans Hsp70‐1 and the effects of polyhistidine tagging on purification yield and ATPase activity
Authors: Odunuga Odutayo O. , Polvadore Elton I. , Choi Ka H. , Bollinger Sarah A.
Published: June 2021

Caenorhabditis elegans has been used as a model organism to study the roles of molecular chaperones in cellular processes. C. elegans heat shock protein 70‐1 gene, CeHsp70‐1, is the first of the 13‐member Hsp70 family genes identified in the organism. The protein product of this gene, CeHsp70‐1, has been shown to play an important role in conferring thermo‐tolerance and longevity on C. elegans. Here, we present the results of the first work to over‐express, purify and characterize the ATP hydrolyzing activity of a member of the C. elegans Hsp70s. Recombinant CeHsp70‐1 was found to be highly expressed and sufficiently soluble in Escherichia coli. The protein was purified to homogeneity using a combination of nickel affinity, ion exchange and size‐exclusion chromatography. Kinetic properties of the basal ATPase activity of the enzyme in a low‐salt buffer were determined using a colorimetric assay. The specific activity, km and kcat values obtained for CeHsp70‐1 were 25 nmol/min/mg, 50 μM and 0.28 min−1, respectively. The kcat of the protein was found to be similar to that of heat shock cognate 70 (Hsc70) and binding immunoglobulin protein (BiP). At low concentrations, CeHsp70‐1 existed mostly in its monomeric form. We show that direct N‐terminal (His) 6‐tagging of CeHsp70‐1 lowers purification yield and affects ATPase activity.
Kinetic analysis of β‐Galactosidase activity from Thermococcus kodakarensis KOD1, an extreme thermophile
Authors: Hwa KuoYuan , Subramani Boopathi , Shen Santai , Lee Yu-May
Published: June 2021

The enzyme β‐galactosidase catalyzes the hydrolysis of lactose into glucose and galactose. An extracellular β‐galactosidase of 489‐amino acid length and 57‐kDa molecular weight monomer was isolated, expressed and purified from an extreme thermophile, Thermococcus kodakarensis KOD1. Here, the enzyme kinetic parameters of the β‐Galactosidase were analyzed through the hydrolysis of the yellow chromogenic substrate, chlorophenol red β‐D‐galactopyranoside (CPRG). Ad, the red colored chlorophenol red product was measured. The optimum pH and optimum temperature for the enzyme activity were 6.5 and 100°C. The maximum enzyme activity was retained even at 110°C, the highest temperature used in the study. The kinetic parameters Km and Vmax of the purified enzyme were 1.4mM and 2.5 U/mg for CPRG at the optimum pH and temperature conditions. The enzyme could not be inhibited with glucosidase inhibitors.

The research was supported by the National Taipei University of Technology to KYH (NTUT 100‐140‐01, NTUT 99‐140‐04) and by Academia Sinica to YML.
ATP alters IKK‐2 inhibitor affinity for IKK2 enzyme and reveals non‐equivalent inhibitor binding sites
Authors: Liu Jia Yeu , Stevens Kirk , Mourey Robert
Published: June 2021

Method

Inhibitor off‐rate was measured by two methods: (1) pre‐binding the enzyme with inhibitor followed by measuring the recovery of enzyme activity upon a 200‐fold dilution of the enzyme‐inhibitor complex in the presence of 1mM ATP and (2) measuring the shift in the compound potency (IC50) curves obtained by prebinding the enzyme and inhibitor in the absence of ATP followed by addition of either 1μM or 1mM ATP.

Results

Three literature inhibitors with potency (IC50) ranging from <1 to 35 nM were used for this study: PHA‐408 (Pfizer), GSK‐082 (GSK), and AVE‐002 (Aventis). The off‐rates (T1/2) measured by the dilution method are 124 and <15 min for PHA‐408 and GSK‐082, respectively. The off‐rate for AVE‐002 was biphasic with half of the enzyme‐inhibitor complex dissociating immediately (T1/2 < 2 min) and the other half bound tightly (T1/2 54 min). The IC50 curves at 1μM and 1mM ATP for PHA‐408 showed no shift using a short 2 min assay and is consistent with the slow off‐rate. GSK‐082 showed a > 10 fold loss of potency with 1mM ATP, as expected for a fast off‐rate inhibitor. AVE‐002 IC50 curve with 1μM ATP was monophasic but displayed a biphasic response with 1mM ATP.

Conclusion

ATP can change the off‐rate of IKK‐2 inhibitor bound to the enzyme. Additionally, high ATP revealed that AVE‐002 bound in the two inhibitor binding sites of the IKK‐2 homodimer non‐equivalently.
Identifying new drug targets against African Trypanosomiasis
Authors: Kalidas Savitha , Phillips Margaret
Published: June 2021

Identifying new drug targets against African Trypanosomiasis Human African Trypanosomiasis (HAT), one of the common tropical neglected diseases (NTDs), is responsible for extensive suffering, death and economic burden in developing countries. NTDs represent the sixth greatest global health burden in terms of the disability adjusted life years. Research and development yielding safe and effective drugs for their treatment and prevention has been recognized as a global health priority. Current drug therapies for HAT are inadequate, hard to administer and have increasing drug resistance problems. To identify and validate new targets for drug discovery we, in a coordinated effort from several groups, are systematically analyzing approximately 200 genes coding for enzymes involved in different biochemical and signaling pathways by RNAi knockdown or gene knockout. We have recently reported the development of a new vector (pTrypRNAiGate) derived from pLEW100 that utilizes the Gateway® recombination system to facilitate easy production of hairpin RNA constructs which facilitates high‐throughput applications for gene silencing and provides a tool for functional genomics in T. brucei. Using this vector we have knocked down 7 aminoacyl tRNA synthetases (AARS), which have an important role in protein translation. RNAi against 5 AARS resulted in growth inhibition of T.brucei bloodstream form in vitro. Two of them, Prolyl tRNA synthetase and Asparagyl tRNA synthetase show morphology changes upon RNAi induction suggesting cell cycle defects. Further validation of AARS, recombinant protein production and assay development is in progress. This work contributes toward a systemic analysis of T. brucei enzymes to identify and validate new drug targets against the infectious disease, African Trypanosomiasis.

NIH Grant: AI078962
A real‐time assay for monitoring Mycobacterium tuberculosis trehalose phosphate phosphatase activity
Authors: Lindenberger Jared J , Lajiness Daniel H , Ronning Donal
Published: June 2021

The otsB2 gene that encodes the enzyme trehalose phosphate phosphatase (TPP) is essential for the viability of Mycobacterium tuberculosis (M. tb). TPP possesses trehalose‐6‐phosphate (T6P) phosphatase activity that is central to the production of trehalose, which acts as the primary mycolate carrier molecule during biosynthesis of the mycobacterial outer membrane (MOM). The essentiality of the otsB2 gene and the role of TPP in trehalose biosynthesis makes TPP an intriguing target for antitubercular drug development. This abstract describes an enzymatic assay that allows real‐time monitoring of the TPP activity and determination of steady‐state kinetic parameters. The assay was analyzed for reproducibility and showed a Z′ value of 0.76 when using T6P at a concentration near the determined Km. The NIH clinical collection was used as a test set to determine assay consistency under typical assay conditions. The results from these experiments gave Z‐factors ranging from 0.44–0.52. Taken together, these data show the assay to be rapid, highly reproducible, and amenable to high‐throughput screening methodologies as a basis for identifying lead compounds that inhibit TPP.
Arachidonic acid inhibits retinoic acid‐induced neuritogenesis of human neuroblastoma SH‐SY5Y cells
Authors: AMISSAH FELIX , AYUK-TAKEM LAMBERT T. , LAMANGO NAZARIUS S.
Published: June 2021

Polyisoprenylated proteins regulate intracellular processes such as cell growth, differentiation, oncogenic transformation and actin cytoskeletal organization. Recent discoveries show that polyunsaturated fatty acids (PUFAs) may be involved in neurodegenerative diseases. PUFAs such as arachidonic (AA), and eicosapentaenoic acids (EPA) induce apoptosis in cultured cells. This effect has recently been shown to correlate with the inhibition of polyisoprenylated methylated protein methyl esterase (PMPMEase). We propose that abnormally high levels of PUFAs may be deleterious to neuronal cells. Enzyme kinetics analysis with PMPMEase revealed a 2‐fold decrease in KM from 16.2 ± 1.34 to 8.60 ± 1.20 and 7.84 ± 1.02 μM in the presence of 10 μM AA or EPA, respectively. A relatively severe degenerative effect of AA on SH‐SY5Y cells was observed in the absence of 10% FBS (EC50 of 6.2 μM). AA (2 – 10 μM) caused concentration and time‐dependent inhibition of neuritogenesis in retinoic acid (RA)‐differentiated SH‐SY5Y cells. Significant decreases in the percentage of neurite‐bearing cells and neurite lengths were observed after 24 h with 10 μM AA. These results suggest that excessive PMPMEase inhibition may extend beyond controlling cell viability to the degeneration of vulnerable cells such as neurons.

Supported by NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020).
Role of Melanocortin 4 Receptor (Mc4R) in Obesity
Authors: Nwana Naderge , Dhir Ravindra , Yin Xaoyan , Ahima Rexford
Published: June 2021

Obesity is increasing worldwide and closely associated with diabetes, abnormal lipid levels, hypertension and other diseases. Melanocortin 4 Receptor (Mc4R) deficiency is the most common monogenic form of obesity. It is estimated that 5% of extremely obese patients have loss‐of‐function mutations of Mc4R. The goal of this project was to determine how Mc4R affects energy homeostasis and diseases associated with obesity. It was hypothesized that a disruption of Mc4R signaling leads to obesity and its associated diseases. Metabolic parameters were compared between normal and Mc4R KO mice. Both genotypes were weaned on standard rodent chow. Food intake was weighed and body composition measured using Nuclear Magnetic Resonance, NMR. At 6 weeks, mice were euthanized and blood was harvested and centrifuged for serum chemistry analysis. Data were analyzed using t test or ANOVA. We found a significant increase in food intake and body weight, in Mc4R deficient mice. There was a mild difference in serum lipid levels suggesting that this model of obesity has a greater adverse impact on glucose homeostasis than lipid metabolism.
BPA leaching in baby bottles upon microwave heating: a simulation of real food conditions
Authors: Tarasenko Alexandra , Benitez Benedetta Sampoli
Published: June 2021

Bisphenol‐A (BPA), commonly found in polycarbonate plastics, has long been suspected to behave as an endocrine disruptor at concentrations well below toxic levels. Recent studies concluded that children drinking from polycarbonate bottles are exposed to enough BPA to cause serious health concerns. Many of the studies use food simulants (such as ethanol) to mimic real foods. However, we employed an ELISA test that can be used directly on different liquids ready for consumption. This study looks at the amount of BPA leached upon microwave heating into milk, infant formula and fruit juices. Previous experiments showed that BPA leached more into water than in milk, suggesting that fat concentration affects the amount of BPA absorbed by the liquid. The samples were tested using different PC containers, including a used PC baby bottle, a used BPA‐free baby bottle and a new PBA‐free baby bottle. The bottle not marketed BPA‐free had leached a significantly higher amount than the now produced BPA‐free bottles, indicating an improvement in the production of polycarbonate bottles. The amount of BPA leaching into actual food will be compared to the leaching into the commonly used simulant for that food to determine if this is an accurate way of testing BPA leaching. This research was supported by the Geraldine Ferraro Center for Educational Excellence in Science, Technology and Mathematics.
Measuring glycogen breakdown pathways using fluorescent derivatives of glucose
Authors: Clifford Patricia , Lundbeck Briana , Bernart Matthew , Parker Glendon
Published: June 2021

A glycogen molecule consists of glucose oligosaccharides and proteins; its structure reveals a branched, densely packed molecule of nearly 50,000 glucose monomers. The breakdown of it occurs through two pathways: 1) a cytosolic mode in which enzymes cleave bonds on the outer branches of the glycogen, 2) simultaneous degradation of the entire molecule in the lysosomes. Both are known to occur within a typical cell; however the instance in which each pathway is initiated has yet to be observed. The purpose of our research is to determine which is employed in response to various environments in the cell. Florescence monitoring of glycogen has previously been hypothesized and two methods are applied in this study: 1) 2‐NBDG (2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diaxol‐4‐yl)amino]‐2‐deoxyglucose), a fluorescent glucose derivative with the ability to be incorporated into the outer non‐reducing terminals of the glycogen, 2) Texas Red‐Hydrazide, which can be incorporated into all glucose molecules following glycogen digestion and glucose oxidation. The Texas Red to 2‐ NBDG ratios can be used to explain the breakdown of the molecule. Glycogen broken‐down gradually in the cytosol generates a large ratio. While glycogen degraded in lysosomes yields a smaller ratio. Pathway activation is assessed under the following conditions: a) ranging glucose and nutrition levels; b) stress (heat, mercury, etc.); c) transfection with RNAi targeted towards glycogen phosphorylase or LAMP‐1 (Lysosomal‐associated membrane protein 1). Understanding the triggers for each pathway will allow for insight into the metabolism of glycogen in response to environmental stimulus.
Micro‐patterned arrays of epidermal growth factor (EGF) reveal stimulated association of paxillin, ERK, and F‐actin with EGF receptors during cell signaling
Authors: Singhai Amit , Bryant Kirsten L , Baird Barbara A , Hammes Stephen R , Holowka David A
Published: June 2021

We are investigating the spatiotemporal association of signaling proteins with EGF receptors (EGFR) using micro‐patterned EGF surfaces. We utilize EGF‐biotin bound to streptavidin that is covalently coupled in ordered micron sized square arrays on silicon surfaces. NIH‐3T3 cells stably over‐expressing wt EGFR are added and adhere to these patterned surfaces. We observe concentration of fluorescently labeled EGFR and stimulated tyrosine phosphorylation that are spatially confined to the patterns of immobilized EGF. We also observe recruitment of paxillin‐GFP to the receptor signaling complexes formed on EGF patterns, and this association occurs in the absence of integrin co‐clustering but depends on EGFR tyrosine kinase activity and F‐actin, which also accumulates in these patterned regions. Inhibition of Src kinase with PP2 inhibits tyrosine phosphorylation of associated paxillin but does not prevent its localization with patterned EGF. We further detect recruitment of phospholipase Cγ‐1‐GFP and phosphorylated Erk to the patterned EGF in a ligand‐dependent manner. Our ongoing studies are investigating the roles of paxillin and F‐actin in recruitment of Erk to the EGF‐mediated signaling complex. These studies highlight the utility of micro‐patterned growth factors to characterize the spatially regulated formation of signaling complexes at the plasma membrane. Supported by NIH Grant R01‐AI018306.
RGS domain‐mediated dimerization regulates GRK5 plasma membrane localization and function
Authors: Xu Hua , Jiang Xiaoshan , Wedegaertner Phillip B.
Published: June 2021

G protein‐coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM) to both terminate further activation of G proteins and promote G protein‐independent signaling. Our goal is to elucidate the mechanisms of PM targeting of GRKs. Despite high sequence similarity, GRK5 and GRK6 constitutively localize at the PM whereas expressed GRK4 is predominantly cytoplasmic. GRK5/GRK4 chimeras and point mutations in GRK5 identified a short sequence within the Regulator of G protein Signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. Interestingly, this region of the RGS domain of GRK5 coincided with a region of GRK6 shown to form a dimeric interface in the published GRK6 crystal structure. Using co‐immunoprecipitation and Bimolecular Fluorescence Complementation assays, results were consistent with GRK5, but not the RGS domain mutant, GRK5‐M165E/F166E, existing as homodimers in cells. In addition, we showed that GRK5‐ M165E/F166E is deficient in inhibiting PAR‐1‐induced calcium release and in promoting neurite extension. The results in this study suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function. Supported by NIH R01 GM56444.
A role of GRK5 in neuronal morphogenesis: promoting actin bundling and targeting bundles to membrane structures
Authors: Wang Feifei , Chen Yuejun , long Hui , Wu Ziyan , Ma lan
Published: June 2021

The extensive membrane remodeling and cytoskeleton dynamics are both required in neuronal morphogenesis, but how these two processes are coordinated spatio‐temporally during neuronal development is largely unknown. Overexpression and depletion studies in neuronal cells indicate that G protein coupled receptor (GPCR) kinase 5 (GRK5), is critical for neuritogenesis, dendrite branching, and maturation of dendritic spines, and the effects are largely independent of its kinase activity. Furthermore, GRK5 promotes F‐actin bundling at membranes of dynamic neuronal structures through interacting with both F‐actin and PI(4,5)P2. Disruption either of the F‐actin and PI(4,5)P2 binding properties of GRK5 results in decreased dendritic arborization and maturation of dendritic spines, indicating GRK5 is not acting as a kinase, but rather provides a key link between the plasma membrane and actin cytoskeleton. Our results reveal that GRK5 functions as a molecular scaffold to promote the formation of actin bundles and targeting them to membranes at dynamic neuronal structures and demonstrate its physiological significance in neuronal morphogenesis.
Expression and Purification of Cdc42 and PBD46
Authors: Evans Edward J , Adams Paul D
Published: June 2021

The Ras superfamily consists of low molecular weight G‐proteins that are heavily involved in cellular signaling, leading to various downstream responses. Like other Ras proteins, Cdc42 acts as a molecular switch interchanging from its active (GTP‐bound) and inactive (GDP‐bound) state through the utilization of GAP (GTPase‐activating proteins) and GEF (guanine nucleotide exchange factors) effectors. It has been shown that Cdc42 signaling pathways play a major role in cell proliferation and differentiation, leading to tumor growth. PBD46 is an effector of Cdc42 proven to inhibit GTP hydrolysis, stabilizing the Ras protein in its active state. PBD46, as well as many other effectors, bind to the Switch I region (amino acids 28–40) of Cdc42. Threonine of position 35 was replaced with alanine forming a Cdc42 mutant. With the use of affinity column chromatography, Cdc42(WT), Cdc42(T35A), and PBD46 were expressed and purified for future tests of the Switch I binding interface.
Gene Expression profile of Staphylococcal Enterotoxin B and Lipopolysaccharide induced human PBMCs
Authors: Mendis chanaka
Published: June 2021

Staphylococcal enterotoxin B (SEB), an exotoxin produced by staphylococcal aureus, is commonly associated with food poisoning and capable of triggering flu‐like symptoms resembling those of endotoxins – such as lipopolysaccharide (LPS) released from Gram‐negative bacterial cell walls. Eventhough both exhibit similar symptoms, each has its unique gene expression profile. Here we attempt to distinguish two toxins that display similar symptoms using multiple gene expression analysis techniques in a time dependent manner. Thirty genes that showed opposite expression patterns when exposed to the two toxins using micro‐array analysis were further analysed using multiple gene quantitation techniques such as RT‐PCR and real time PCR. These expression patterns were further validated by investigating the protein expression pattern. We believe that this study will not only allow us to confirm the results obtained through micro‐array experiments but also to better predict, identify and isolate varies exposures in a rapid and an accurate manner.
AN INTEGRATED EXPERIMENTAL WORKFLOW TO INCREASE THROUGHPUT AND DATA ROBUSTNESS FOR ANALYSIS OF MAMMALIAN PROTEIN INTERACTION NETWORKS
Authors: Auerbach Daniel , Bruderer Roland , Uhlmann Thomas , Rezwan Mandana
Published: June 2021

To understand the organization of protein complexes and how biological information is propagated via protein‐protein interaction is becoming increasingly important to get new functional insights on cellular processes. Affinity purification coupled to mass spectrometry (AP‐MS) has been successfully used in the past to characterize protein complexes. However, mainly due to significant experimental limitations the success and power of the AP‐MS strategy has been underrated to date. Here, we describe an integrated workflow for characterization of mammalian protein complexes. The experimental pipeline comprises rapid generation of isogenic mammalian cell lines, protein complex analysis using an efficient double affinity purification strategy followed by state‐of‐the‐art nano‐LC‐Orbitrap‐MS analysis.

We use this pipeline to study interactions associated with BMP signaling systems. In particular we focus on ACTR‐1/Alk2 and FKBP12; two proteins intimately linked to Fibrodysplasia Ossificans Progressiva (FOP) a catastrophic human disorder leading to heterotopic ossification throughout the body.

The high performance of our strategy is well suited to identifying protein interactions partners that can subsequently be used as (1) entry points for focused research projects and (2) to more globally map out whole signaling systems linked to important biological processes.
Proteomic profiling of chemoresistance in pancreatic adenocarcinoma
Authors: Hwang Sun-il , McKinney Kimberly Q , Truong Tam , Lee Yong-yook , Sindram David
Published: June 2021

Gemcitabine‐based chemotherapy is offered to patients with pancreatic adenocarcinoma (PC) per current clinical guidelines, with minimal benefit to disease free and overall survival due to rapid development of chemoresistance. Our objective in this study was to identify proteins involved in chemoresistance using proteomic profiling of GEM resistant (Panc‐1, MiaPaCa‐2) and GEM sensitive (BXPC‐3, Su8686) PC cell lines. Total protein lysates were prepared from the cell lines, separated by SDS‐PAGE and digested using standard protocols. Tryptic peptides were analyzed in duplicate on the mass spectrometer. Spectra were searched against the IPI protein database using the Sequest algorithm, then analyzed using Scaffold software and Power Law Global Error Model. We identified 19 proteins with statistically (p<0.005) increased expression in drug resistance, and 57 proteins decreased in drug resistant cells. Various proteins known to be involved in epithelial to mesenchymal transition, which is associated with increased chemoresistance and progression of PC, are differentially expressed in the cell lines. We validated our findings using western blot. We conclude that proteomics can be used to identify chemoresistance related proteins, a crucial step in combating drug resistance in PC treatment.
Use of AFLP to study the genetic variability of Leishmania sp
Authors: Restrepo Carlos M , Lao Efraín Pérez , De La Guardia Carolina , Sousa Octavio E , Calzada José E , Lleonart Ricardo
Published: June 2021

AFLP is a very efficient technique for rapid detection of genetic variability, particularly useful on organisms without sequenced genomes. The objective of this study was to assess the amplified fragment length polymorphisms (AFLP) for testing genetic diversity in Leishmania sp. Leishmania species used for this study were representative of the Viannia and Leishmania subgenus. Fluorescent AFLP kits (Applied Biosystems, USA) were used for the whole procedure. Detection and sizing of peaks were conducted with GeneMarker v. 1.91 software (Softgenetics, USA). Then error rate was estimated for every possible pair of replicates using ten replicates from a single L. panamensis promastigotes culture. We have found that this technique is able to generate high numbers of peaks when low selective EcoRI and MseI primers were used (+0, +1, +2 series). Additionally, we have found that up to 86,9% of the primer combinations are polymorphic. Several fragments were apparently specific for L panamensis, L. (Viannia) and L. (Leishmania). The error rates estimated from every possible pair of replicates ranged from 0% to 6%. Cluster analysis results are in agreement with the current known taxonomy of Leishmania species. In conclusion, AFLP is a molecular fingerprinting technique showing promising potential to explore genetic diversity at Leishmania sp., both at intraspecific and interspecific levels. Its application will allow researchers to look deeper into new polymorphisms and possibly correlate them to ecoepidemiologically relevant traits and other important aspects of the biology of the parasite.
Computational Analysis between two different hemoglobins from Caspian Sea sturgeons (Acipenser persicus and Acipenser stellatus) induced by n‐dodecyl trimethylammonium bromide
Authors: Ariaeenejad Shohreh , Jamili Shahla , Rezaei Mehran Habibi , Fatemi Mohammad Reza , Poursasan Najmeh , Kavousi Kaveh , Moosavi-Movahedi Ali A
Published: June 2021

The multiplicity of the Hemoglobins (Hbs) of the and the A. stellatus, two species from the Caspian Sea, A.persicus was confirmed with the polyacrylamide gel electrophoresis (SDS‐PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) experiments. In the current study, the interaction of n‐dodecyl trimethylammonium bromide (DTAB) as a cationic surfactant under variable concentrations, with the purified dominant Hbs was investigated using UV‐visible absorption spectra and circular dichroism (CD). The chemometric resolution techniques were used to determining the number of the components and mole fraction of the oxidized Hbs. The analysis of the obtained spectral data using singular value decomposition (SVD), evolving factor analysis (EFA) and multivariate curve resolution‐alternative least square (MCR‐ALS) as well‐known chemometrical techniques, gave the evidence for the existence of four different molecular components including native (N), intermediate (I), intermediate (II) and denatured (D) in sturgeon Hbs. On the basis of the interpretation of chemometrical results, the formation of intermediate states of fish Hbs, plays an important role for regulation of the respiratory system of the fishes proportional to their habitat depths and environmental partial pressure of oxygen.
Development of Pattern Recognition Algorithms for Identification of Gut Microorganisms using Fatty Acid Signatures
Authors: Shen Frank , Ballesteros Dora , Sasser Myron , Cimini Len , Dhurjati Prasad
Published: June 2021

A pattern recognition algorithm was developed that was capable of accurately identifying the presence of specific microbial species in gut samples from the sample's composite fatty acid signature. We focused on 51 different species found in the human gut and 61 different fatty acids produced by those species. The unique fatty acid signatures of the individual microorganisms were obtained by gas chromatography (provided by MIDI Inc). Several algorithms were developed in Matlab to differentiate individual species using the fatty acid composition data of the simulated mixtures. These algorithms utilized Hamming distance, least‐squares optimization, and combinatorics methods to identify the presence of each species. The algorithms were tested against myriad virtual samples to analyze their ability to detect the presence of selected species. While more complex mixtures of species decreased the probability of detection, we found that utilizing a constrained least‐squares method with Hamming‐based filters provided high probabilities of detection for all tested samples. This algorithm was capable of detecting the presence of a selected species over 90% of the time for instrument tolerances as high as 15%.
Use of phylogenetics for the purpose of quality control in the green algae genus Chlorella
Authors: Jensen Travis Lee , Stubbendeck Reed M. , Barnes Austin L. , Wichers Katie M. , Keller Bryant J. , Bailey Cheryl A.
Published: June 2021

The push for an efficient biofuel is at an all time high all around the world. Lately, a consideration for an algal biofuel has made algae a prime candidate for research. However, the most efficient way to obtain a known culture is to receive it form a culture collection. Or is it? Our research has shown that the genus Chlorella has some issues according to the DNA of the organisms compared to the taxonomy of the culture collections. To show the differences of what the cultures are compared what they are said to be, the pure culture was isolated, its DNA was extracted, PCR amplified, and sent to sequencing. Using the MEGA 5 program in association with the (Internal Transcribed Spacer) ITS2 database, the sequence is annotated. Each element (18S, ITS1, 5.8S, and ITS2) at this point is individually aligned. The elements are then concatenated, and a maximum likelihood tree is run on the MEGA 5 program. This produces a tree that species in the Chlorella genus will group together in an obvious way with acceptable bootstrap values. With this tree, it will be obvious which strains belong to a given species, and which do not. The source of support for this project comes from NSF 25‐6230‐0130‐009
Regulation of I‐kappa‐B kinase (IKK) Pathway by CARMA 1•Bcl‐10•MALT‐1 (CBM) Complex Promotes SNARE Complex Formation and Secretion in Platelets
Authors: Banerjee Meenakshi , Karim Zubair A. , Zhang Jinchao , Whiteheart Sidney W.
Published: June 2021

In deciphering the underlying mechanisms of how the platelet SNARE complex machinery modulates thrombosis and granule release, we have discovered that agonist‐dependent stimulation of platelets leads to the phosphorylation of the t‐SNARE, SNAP‐23, at Ser95, by I‐kappa‐B Kinase (IKK) thus playing a key role in SNARE complex assembly and secretion. The presence of the IKK complex (IKKα, IKKβ, IKKγ) and its agonist‐dependent phosphorylation, in anucleate platelets implies for a unique non‐transcriptional activity. Inhibition of IKK using inhibitors showed that both SNAP‐23 phosphorylation and platelet secretion were affected without adversely affecting calcium mobilization, cytoskeletal changes, or tyrosine kinase signaling. Corroborating these studies, in vivo studies in mice treated with IKK inhibitors significantly prolong tail bleeding times confirming secretion defect. Further analysis of the upstream elements of the pathway suggests that CARMA1‐Bcl10‐MALT‐1 (CBM) proteins are present and their differential translocation to lipid rafts, which abound in SNAP‐23, upon agonist stimulation in platelets emphasizes the novel regulation of the CBM‐IKK‐SNAP‐23 pathway in promoting SNARE complex formation, platelet exocytosis and possibly in mediating platelet inflammatory responses. Collectively these data implicate CBM‐IKK‐SNAP‐23 signaling cascade in controlling platelet thrombosis with long‐term studies aimed at developing these as anti‐thrombotic therapeutics. (Supported by HL56652 and HL091893 to S. W. W)
Time‐dependent changes in long range sphingolipid organization revealed by high‐resolution secondary ion mass spectrometry
Authors: Kraft Mary L , Klitzing Haley A. , Lou Kaiyan , Zimmerberg Joshua , Weber Peter K.
Published: June 2021

To coordinate cell signaling and other membrane‐mediated cellular functions, the cellular plasma membrane is organized into domains of differing protein and lipid composition. Though the distributions of specific membrane proteins can be imaged with many techniques, the precise spatial organization of sphingolipids within the plasma membrane is not well‐established. Here we have combined metabolic isotope incorporation with high‐resolution secondary ion mass spectrometry (SIMS) to chemically image the distributions of sphingolipids within the plasma membranes of intact fibroblast cells with at least 100 nm lateral resolution. Using this approach, we characterize how the sphingolipid distribution changes as a function of metabolic pulse‐labeling time. We show that changes in the short range (<1 micron) sphingolipid organization are not detectable, but the non‐random sphingolipid organization occurring on longer length scales (>5 microns) varies with the duration of selective isotope incorporation.
A new functional role of potassium
Authors: wu xiaobin , Lee Jaekwon , Kim Heejeong
Published: June 2021

Metal ions play vital roles in numerous biological processes, including energy generation, neuro‐transmission, signal transduction, transportation of nutrients, and maintenance of pH and osmolarity. To gain better insights into metal metabolism and metalloprotein biosynthesis, we have searched new genes involved in copper and iron utilization. A predicted potassium‐proton antiporter was identified as a new molecular factor necessary for biosynthesis of functional ferroxidases at the secretory pathway. Copper serves as a cofactor of ferroxidases in various organisms, including humans. Both copper and iron are required for respiratory chain electron transport and many other vital biochemical processes; however, the implication of potassium in copper and iron metabolism is an unanticipated discovery. Several lines of our study indicate that potassium transported into the secretory pathway is required for copper metallation of ferroxidases during their biosynthesis. This new discovery has opened avenues by which we can gain better insights into the mechanisms underlying biosynthesis of cuproproteins and potentially other metalloproteins. This study characterizes a new molecular factor involved in the metabolism of vital metal ions, copper, iron and potassium and discovers a novel functional role of a major intracellular cation potassium.
Genetic determinants of human serum sterol levels
Authors: Stiles Ashlee Renee , Russell David W
Published: June 2021

24‐hydroxycholesterol (24‐OHC) is a metabolite of brain cholesterol metabolism and a substrate for bile acid synthesis. Mutation of the cholesterol 24‐hydroxlase gene in mice (Cyp46A1) causes severe learning defects. Targeted and untargeted approaches were taken to identify genetic determinants controlling serum 24‐OHC levels. First, we measured 24‐OHC levels in 3230 individuals from the Dallas Heart Study, a multi‐ethnic patient cohort. Genome‐wide analysis of non‐synonymous variations and dense single nucleotide polymorphism (SNP) scans of lipid metabolism genes revealed one highly significant (P=2.1× 10−27) genetic association between elevated 24‐OHC and SNPs in an oxysterol 7□‐hydroxylase gene (CYP39A1). Expression analysis in transfected cells revealed that the identified SNPs decreased CYP39A1 enzymatic activity when compared to wild‐type, revealing a loss of enzymatic function. Currently, we are correlating these findings with clinical phenotype data to reveal the role of this oxysterol in disease which allows others to understand the roles of oxysterols in‐vivo. Similar association studies were done for other sterols measured in the cohort and results from these experiments will be reported.
Differential regulation of Rap1 through bioactive lipid production in the human platelet
Authors: Holinstat Michael , Yeung Jennifer , Vesci Joanne , Arnouk Alex , Ikea Kenneth , Holman Theodore R
Published: June 2021

Platelet activation plays a crucial role in hemostasis and thrombosis and a central role in the pathophysiology of cardiovascular disorders. Activation of the small G protein Rap1 is essential for integrin activation leading to platelet aggregation. As bioactive lipids play an important role in regulating platelet reactivity, we hypothesize that regulating the fatty acid substrates available for oxidation by COX‐1 and 12‐LOX will directly affect Rap1 activity and platelet activation. Therefore, platelets were treated with several polyunsaturated fatty acids (PUFAs) or their oxidized metabolites and thrombin‐induced Rap1 activation was measured. Rap1 activation was inhibited in the presence of the PUFAs DGLA and DHA, but not EPA or ALA. Similar inhibition of Rap1 activation was observed in the presence of the metabolites for each PUFA tested. Additionally, integrin activation and platelet aggregation were inhibited under similar conditions. Finally calcium mobilization and PKC activation, known regulators of Rap1 activation, were measured in the presence or absence of PUFAs and found to be inhibited by some, but not all metabolites. This study supports an important role for the relative content and oxidation of PUFAs on the membrane by COX‐1 and 12‐LOX in regulating the level of Rap1 activity, platelet reactivity, and clot formation in human platelets. This study was supported by NIH grant HL089457.
The regulation of thrombosis and hemostasis by fatty acid metabolites
Authors: Yeung Jennifer , Arnouk Alex , Vesci Joanne , Ikei Kenneth , Holman Theodore R. , Holinstat Michael
Published: June 2021

Excessive platelet activation often results in unwanted clot formation leading to vessel occlusion and stroke. One approach to preventing clot formation in the vessel may be through regulation of signaling events in the platelet. 12‐lipoxygenase (12‐LOX), an enzyme regulating bioactive metabolite formation in human platelets, oxidizes a number of free fatty acids such as arachidonic acid (AA) and dihomo‐γ‐linolenic acid (DGLA) following platelet activation and fatty acid liberation from the phospholipid membrane. We hypothesized the metabolites formed by each fatty acid may have opposing regulatory effects in the platelet. To investigate this possibility, a number of biochemical endpoints were assessed in the absence or presence of AA, DGLA, or their metabolites (12‐HETE and 12‐HETrE, respectively). Washed human platelets treated with DGLA or 12‐HETrE significantly attenuated agonist‐induced platelet aggregation, αIIbβ3 activation, and Rap1 activation; whereas AA and 12‐HETE had no inhibitory effect on platelet‐mediated activation. Our data supports an inhibitory role for 12‐HETrE and a pro‐thrombotic role for 12‐HETE in the platelet. Hence regulation of fatty acid content or metabolite formation may represent alternative mechanisms for regulating platelet reactivity in vivo. This work was supported by Sigma Xi Grants‐in‐Aid and NIH grant HL089457 (to MH).