bluuebunny/crossref_metadata_2025 huggingface dataset:
Replacing animal models of osteoarthritis with 3‐dimensional models of articular cartilage and synovium
Authors: Mobasheri Ali , Shakesheff Kevin , Shakibaei Mehdi
Published: June 2021

Osteoarthritis (OA) is an increasingly common clinical problem in ageing humans and companion animals. It affects load‐bearing synovial joints causing pain, inflammation, loss of mobility and significant morbidity. A number of animal models of OA are currently used to study disease pathogenesis and evaluate the efficacy of candidate anti‐inflammatory and anti‐arthritic drugs for clinical development. These animal models may involve injection of inflammatory agents into the joint, surgical creation of joint instability or surgical replication of joint trauma. Such models not only cause a considerable amount of pain and suffering but also none of them have a proven track record of predictability in human disease. Consequently, there is an acute need for developing novel and alternative in vitro models that closely mimic the disease. We have used articular cartilage and synovial tissues from horses and dogs euthanased for unrelated clinical reasons to establish several in vitro models of the joint. We have developed an explant model of articular cartilage inflammation, co‐cultures of primary synoviocytes and chondrocytes as well as 3‐dimensional cultures of chondrocytes and mesenchymal stem cells (MSCs). These approaches will help reduce the number of laboratory animals used in OA research and may provide a realistic alternative to experimental animals.
A study of PON1 as a susceptibility gene for hyperlipidaemia and onset of coronary artery disease
Authors: Muiya Paul , Wakil Salma , Al‐Najai Mohammed , Vigilla Mary Grace , Andres Editha , Alshahid Maie , Dzimiri Nduna
Published: June 2021

The involvement of paraoxonase 1 (PON1) gene polymorphism in hyperlipidaemia (HPLD) and early onset of coronary artery disease (CAD) remains poorly studied. In a preliminary genome‐wide linkage study of early onset CAD involving two families (22 individuals) with predominant familial hypercholesterolaemia, we identified several peaks, among others, on chromosome (chr) 7 which harbours the PON1 gene using the Affymetrix Gene Chip 250 sty1 mapping array. The present study pursued a population‐based association analysis for 6 selected SNPs rs854560 (1), rs662(3), rs854552 (3), rs3917577 (4) and rs3735590 (5) in the PON1 gene in 3048 individuals (1013 HLPD cases versus 2035 controls) using the Applied Biosystems real‐time PCR procedure. Two of the 6 variants, rs662 [Odds ratio(95% Confidence Interval = 1.13(1.01–1.26); p=0.031] and rs3917577[1.20(1.04–1.38); p=0.013], individually conferred risk for HLPD. While no association was found between any of these variants with CAD (1796 cases versus 1261 controls) per se, classifying the CAD patients as harbouring (n=748) or not harbouring HLPD (n=997) retained the significance of the rs3917577 as a causative factor [1.23(1.03–1.47); p=0.023] for CAD. In contrast, classifying the HPLD patients as having CAD or not, yielded no delineable relationship. On the other hand, the rs854552 was associated with resistance (p=0.007) to developing HPLD among the CAD individuals. No confounding effect of gender, age, MI, T2D, or obesity was observed on these relationships. The results indicate the PON1 gene is a susceptibility locus for HLPD, and harbouring of the rs3917577 variant increases the risk of acquiring CAD among hyperlipidaemic individuals.
Modeling botulinum neurotoxin type A using 3D printing technology
Authors: Nguyen‐Parsons MyLinh , Collier Rachel , Douglas Cydney , Esbensen Laura , Nguyen‐Parsons MyLe , Wylie Kiersti , Nguyen‐Parsons Lan , Luttgen Madelyn , Thompson Aaron , Stevens Raymond , Kuhn Peter
Published: June 2021

The Clostridium botulinum bacteria produce 7 different neurotoxin serotypes (types A–G) that cause botulism, and these are the most toxic proteins known to man. Botulism develops if a person ingests the toxin, or if the Clostridium bacteria grow in the intestines or wounds in the host and toxin is released. The toxin paralyzes victims by blocking the nerve's ability to communicate with muscle fibers by inhibiting the release of acetylcholine at the synaptic clefts of neuromuscular junctions. The BoNT/A crystal structure solved by the Stevens Lab revealed three domains that are involved in catalytic cleavage of SNARE proteins, translocation of the catalytic domain, and cell receptor binding (Lacy and Stevens 1998). BoNT/A's zinc‐dependant catalytic domain cuts and disables SNAP‐25, one of the major SNARE fusion proteins required for acetylcholine vesicle docking to the presynaptic membrane. Without acetylcholine to properly signal muscle contraction, breathing muscles become paralyzed and the victim eventually suffocates. Although the potential use of BoNT/A as a bioweapon is a major concern, it is also effectively used for numerous therapeutic and cosmetic purposes. The North San Diego Girls Cooperative SMART Team (Students Modeling A Research Topic) developed a model of BoNT/A using 3D printing technology.
The rs2228671C>T of the LDLR gene confers risk for low HDL and early onset of coronary artery disease
Authors: Alrasheed Maha M , Muiya Paul , Wakil Salma , Al‐Najai Mohammed , Andres Editha , Mazher Nejat , Dzimiri Nduna
Published: June 2021

The role of the low density lipoprotein receptor (LDLR) gene polymorphism in hyperlipidaemia (HPLD) and early onset of coronary artery disease (CAD) is not fully understood yet. In a preliminary genome‐wide linkage study in a family of 11 individuals with severe familial hypercholesterolaemia and the index case having undergone triple coronary artery bypass surgery at 14 years, we identified potential loci on chromosome 19, which harbours the LDLR gene. We then sequenced the LDLR gene in all family members and discovered 11 SNPs, for which the proband uniquely harboured homozygous mutant allele for all. In contrast, the only normolipidaemic sibling for all 3 lipid levels carried the homozygous wild‐type alleles, except for the rs2228671C>T (p.Cys27Thr) and one novel SNP for which he was heterozygous. Interestingly, the mother and other carriers of C/C (p.Cyc/Cys) genotype (3 affected and 1 unaffected siblings) also harboured low‐to‐borderline HDL‐cholesterol (HDL‐C) levels. To test the general applicability of this phenomenon, we genotyped 70 individuals with low HDL‐C from the general population. The study showed that 52(73%) were homozygous for the C allele, 17(24.3%) were C/T and only 1(1.4%) carried the T/T genotype, unequivocally linking the p.Cyc/Cyc with the presence of low HDL‐C levels. Our results demonstrate that the LDLR is a susceptibility gene for early onset of CAD and the rs2228671C>T confers risk for acquiring low HDL.
Mechanisms of Aldosterone Action on NCC
Authors: Ko Benjamin , Moddes Lauren , Cooke Leslie , Hoover Robert S
Published: June 2021

Aldosterone increases NCC function and protein expression in whole animal studies at 72 hours but more acute effects of aldosterone on NCC activity have not been studied. We have recently demonstrated that in a mammalian cell model with robust native NCC activity (mDCT15), aldosterone stimulated NCC activity by 26% at 6 hrs and by 52% at 24 hrs. However, the mechanism by which this acute stimulation was mediated is unknown. To examine this question, mDCT15 cells were incubated with either 100nM aldosterone or vehicle (DMSO) for 6 or 24 hours. Cells were then surface biotinylated and surface expression was assessed by Western blotting. Total NCC protein expression remained unchanged compared to control in both the 6 and 24 hour treatment groups. Furthermore, NCC surface expression did not change in the 6 or 24 hour aldosterone treated groups compared to control. This suggests that the effects of aldosterone upon NCC are not primarily mediated by increased surface expression, but instead due to an increase in individual transporter activity. Further work is needed to characterize this mechanism.

Research support: NIH‐NIDDK K08 DK070668, DK081728.
CCL23 upregulates expression of Epidermal Growth factor Receptor and inhibits EGF‐induced proliferation of endothelial cells
Authors: Kim Jee Young , Kwon Taek Hwan , Han Kyu Yeon , Lee Tae Hoon , Kim Jiyoung
Published: June 2021

It has been recently reported that human CCL23 (also known as MPIF‐1, MIP3, or CKβ8) promotes endothelial cell migration and tube formation, while it has no effect on proliferation of endothelial cells. In this study, we conducted DNA microarray analysis to determine gene expression alterations in response to the treatment of HUVECs with CCL23 using 22,000 human genes microarray. We found that the CCL23‐dependent modulation in the expression of genes represented a wide variety of functional categories including genes important in the cell growth and maintenance. Based on the microarray analysis data, we focused on up‐regulation of Epidermal Growth Factor Receptor (EGFR) by CCL23, which was identified by reverse transcription‐polymerase chain reaction, quantitative real‐time polymerase chain reaction and Western blot. EGF stimulated proliferation of HUVECs, which was significantly inhibited by pretreatment of CCL23 in dose‐dependent manner. EGF‐stimulated phosphorylation of ERK was reduced in CCL23‐treated cells, while phosphorylation of STAT1 was increased by CCL23, indicating that STAT1 is involved in the inhibition of EGF‐induced proliferation of endothelial cells by CCL23.

This research is supported by a grant from National Research Foundation of Korea.
Role of KLF2 in endocardial cushion development in mice
Authors: Haar Jack L , Chiplunkar Aditi R , Lung Tina K , Lloyd Joyce A
Published: June 2021

Krüppel‐like factor 2 (KLF2) belongs to a family of transcription factors that have 3 C2/H2 zinc finger domains. KLF2 is expressed in endothelial cells and is induced by shear stress. KLF2−/− embryos in FVB/N genetic background die by embryonic day 11.5 (E11.5) (chi‐square p = 0.0044), whereas in mixed genetic background, they die by E14.5. Our studies show that unlike WT hearts, in E9.5 KLF2 KO hearts (n = 3) the cells lining the atrioventricular (AV) cushion, the area that will become the AV valves, form multiple, disorganized layers and the number of cells is 2‐fold higher. Electron microscopy revealed that compared to WT, the E9.5 KLF2−/− AV canal is narrower in some locations but patent and lined by endothelial cells having numerous cytoplasmic processes extending toward the lumen. In E10.5 KLF2−/− embryos (n = 3), the AV and outflow tract cushions are hypoplastic and the cells lining the AV canal form multiple disorganized layers. Alcian blue staining of E10.5 KLF2−/− hearts showed an absence of glycosaminoglycans in the cardiac jelly. These findings suggest that KLF2, directly or indirectly, regulates endothelial to mesenchymal transformation in AV cushion development. The AV phenotype is observed in FVB/N KLF2−/− embryos but not in embryos of mixed background (n = 2). Therefore, KLF2 is required for normal endocardial cushion development in FVB/N genetic background mice. (Supported by NIH R01DK074694)
Isolation and Purification of Actin Homolog MreB from Caulobacter crescentus
Authors: Currington Rashalai A , Klein Eric , Gitai Zemer
Published: June 2021

Caulobacter crescentus is an important model for studying cell cycle regulation, cellular differentiation, and cell polarity. The perceived absence of a cytoskeletal network in bacteria was once considered a defining distinction between prokaryotic and eukaryotic cells. However, orhtologues of all three major classes of eukaryotic cytoskeletal proteins have been identified: FtsZ, a tubulin homolog, MreB and ParM, actin homologs, and CreS (crescentin) which appears to be an intermediate filament protein. MreB is required for the maintenance of the rod cell shape. MreB is organized in an axial spiral that is dynamically rearranged during the cell cycle. Little is known about the mechanisms regulating MreB polymerization. To this end, we aimed to purify MreB for use in in vitro polymerization assays. We used two schemes to purify C. crescentus MreB. The first method employs a cleavable GST‐MreB fusion protein overexpressed in E. coli. The second method takes advantage of buffer conditions that support either polymerization or depolymerization of MreB: MreB transitions between soluble and insoluble states, with purification by ultracentrifugation. Elucidation of MreB polymerization and depolymerization mechanisms has the potential to impact the design of chemotherapeutics affecting cell cycle regulation, cellular differentiation, and cell polarity. Supported by NIH T34GM008411
A promiscuous ubiquitin ligase controls cell cycle through a unique mechanism
Authors: Glenn Kevin Andrew , Wen Hsiang M. , Kim Namhun , Mallinger Adam , Gonzalez‐Alegre Pedro
Published: June 2021

Objective

Identify the cullin partner proteins for FBG1.

Methods

Western blots of proteins co‐immunoprecipatating with FBG1 identified partner proteins. Growth rates determined by replicate cell counting via a hemocytometer or Countess automated counter. Cell cycle analysis was by FACS.

During cell proliferation, protein degradation is strictly regulated by two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F‐box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF complexes degrade proteins during the G1 phase, but as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. This study explores an unexpected interaction between APC‐2 and SCFFBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Site‐directed mutagenesis shows that this interaction requires a D‐Box within FBG1. Unexpectedly, we demonstrate that co‐expression with FBG1 increases total APC2 levels. Finally, FACS analysis demonstrates that FBG1 induces S‐phase arrest, illustrating the functional consequences of this interaction. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent of its ubiquitin ligase function.

Funding: V.A. RCD and Ellison New Scholars in Aging Award.
Genetic Characterization and Analysis of Cis and Trans‐elements That Facilitate Genome Stability in Saccharomyces cerevisiae
Authors: Jones Hope , Paek Andrew , Weinert Ted
Published: June 2021

Chromosomal fragile sites (CFS's) are specific loci associated with a high frequency of breakage and recombination. A cell's ability to repair and/or replicate through a lesion is prerequisite to the maintenance of genomic stability. This work seeks to identify and characterize both trans and cis CFS associated elements involved in instability onset and progression. An array of S. cerevisiae isogenic DNA repair deficient mutants were utilized to identify genes contributing to the stability or instability of a natural CFS ~ 403 kb from the left telomere on chrVII. Findings suggest that the RAD52 epistasis group, MRX, NHEJ, MUS81 and SGS1, TLS polymerases, and a majority of PRR proteins are required for faithful replication of the 403 CFS, and likely other CFS's as well. In contrast, we find that MMS2 (previously thought to be specific to the PRR pathway), MGS1 (homolog of human WHIP), and POL3 (DNA pol delta subunit) promote error prone events at regions at risk of replication damage. We further find the presence of inverted repeats (IR) are sufficient to induce instability. Two IR's proximal to the 403 site consistently fuse to generate acentric and dicentric chromosomes involving the 403 CFS and a newly identified site on chrVII as well. The frequency of fusion events is aggravated by chromatin traffic stressors such as tRNA transcription induced fork stalling and replisome termination regions.
An analytical mass spectrometry method of determining binding constants between chemokine, posttranslationally modified chemokine receptor N‐terminal peptides, and glycosaminoglycan
Authors: Jen Connie H. , Leary Julie A.
Published: June 2021

Chemokines play roles in inducing chemotaxis, extravasation, and activation of leukocytes. Tyrosine sulfation of the chemokine receptor is crucial for binding to its ligands and signaling. A robust analytical method for measuring the contribution of this posttranslational modification to the receptor to binding of its ligand is lacking. Using Nano‐electrospray time‐of‐flight mass spectrometry, we determined the association constant of chemokine, CCL7 to variably sulfated CCR2 peptides. Association constants between CCL7‐CCR2, CCL7‐monosulfated CCR2, and CCL7‐ disulfated CCR2 were determined to be 1.1 x 104 M−1, 3.9 x 104 M−1, and 4.0 x 105 M−1, respectively. Chemokines also bind to glycosaminoglycans (GAGs). Mutation studies suggest a partial overlap between the GAG and receptor binding domains, but it has been debatable whether a ternary complex forms. Using mass spectrometry we did not observe significant ternary complex formation. Thus, competitive binding experiments between Arixtra and disulfated CCR2 were conducted. As the concentration of disulfated CCR2 is increased from 0μM to 10μM two observations are made suggesting that CCR2 competes with GAG for CCL7 binding: 1. more noncovalent complexes between CCL7 and CCR2; 2. Less noncovalent interactions between CCL7 and Arixtra.
Phylogenetic analysis of the domains that conform the NADPH oxidases
Authors: Julián‐Sánchez Adriana , Magis‐Weinberg Lucía , Riveros‐Rosas Héctor
Published: June 2021

NADPH oxidases (Nox) are eukaryotic enzymes used deliberately to produce superoxide radicals. In a previous work (Mol. Microbiol (2003) 50: 1241), we show evidence that suggests that Nox evolutionarily derived from ferric reductases, which have been described in animals, plants, fungi and bacteria. Both are membrane proteins and contain a cytochrome b as well as flavin, and possess three different domains: a FAD binding domain, a NADP+ binding domain, and a ferric reductase domain. In this work a phylogenetic independent analysis was made for each one of these domains, to obtain additional insight about the origin of Nox.

The analysis was carried out in silico, starting from the protein sequences obtained on public databases (PFAM, NCBI and SwissProt), that possess the NADP+ binding domain (565 sequences), FAD binding domain (607 sequences) and ferric reductase domain (1012 sequences). The domain alignments were built with Clustal_X (corrected manually with BLASP results). Phylogenetic analysis was performed in MEGA 4.0. The results show that in the three analyzed domain, all the Nox comprise a monophyletic group; however, its relationship with the ferric reductases (the proposed ancestral group) is not so clear when the domains in an independent way are analyzed. This contrast to the obtained results with the complete sequences. Supported partially by DGAPA‐UNAM grant IN208510‐3.
AN INTEGRATED EXPERIMENTAL WORKFLOW TO INCREASE THROUGHPUT AND DATA ROBUSTNESS FOR ANALYSIS OF MAMMALIAN PROTEIN INTERACTION NETWORKS
Authors: Auerbach Daniel , Lentze Nicolas , Lampart Thomas , Gstaiger Matthias , Aebersold Ruedi , Glatter Timo
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 have used the system to study interactions associated with the human protein phosphatase 2A and NF‐kB systems. Analysis of these systems revealed new components of protein network structures that are linked to a number of important biological processes such as transcription, the cell proliferation, apoptosis and inflammation. 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.
A molecular car‐crash: a speeding motor hits a new ultra‐stable non‐covalent interaction
Authors: Chivers Claire Elizabeth , Crozat Estelle , Chu Calvin , Moy Vincent , Sherratt David , Howarth Mark
Published: June 2021

Streptavidin is used widely in biotechnology because of the exceptional stability of its binding to biotin. This stability is, however, limiting for applications such as imaging and nanotechnology. We engineered a mutant streptavidin with much slower dissociation from biotin. This mutant streptavidin also had increased mechanical strength and thermostability. We applied this mutant to analyze force generation by FtsK, one of the fastest known molecular motors. FtsK displaced proteins bound in its path and induced rapidly the release of streptavidin from biotinylated DNA. On the other hand, the mutant streptavidin resisted displacement, indicating the force generated by FtsK translocation. The resilience of the binding of this mutant streptavidin may find many applications in biotechnology and biophysics. Funding was provided by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council Chemical Biology DPhil programme and Somerville College Oxford.
Signal perception and transmission in histidine autokinases: Insights from the Agrobacterium tumefaciens VirA/VirG system
Authors: Lin Yi‐Han , Gao Rong , Binns Andrew N , Lynn David G
Published: June 2021

VirA, the histidine autokinase found in Agrobacterium tumefaciens, is activated in response to several signaling cues, among which the plant‐derived phenolic molecule being the most critical. The cytoplasmic linker domain is responsible for phenol sensing, and a coiled‐coil was predicted to bridge the linker domain to the kinase. Using yeast transcription factor GCN4, the possible signal‐induced rotational motion in the coiled‐coil was examined. Through different insertions between GCN4 and the coiled‐coil, the signal‐independent ratcheted activities were observed. Random mutagenesis and functional library screening also indicated several constitutively on mutants in the coiled‐coil, which enabled the dimerization interface to be established. Together with the structure model of the linker domain, generated by homology modeling, and the possible phenol binding site, probed by computational docking, the model for phenol perception and the associated motion in the signaling module of VirA is proposed.

This work is supported by NSF‐IOS‐0818613.
Human Embryonic Stem Cells Maintain Pluripotency after E‐Cadherin Expression Knockdown
Authors: Byas Shanel Nicole , Klim Joseph , Kiessling Laura
Published: June 2021

E‐cadherin is a Ca2+‐dependent adhesion molecule essential for cell‐cell interaction [1]. E‐cadherin has been reported to have a crucial role in cellular migration, proliferation, differentiation, and the maintenance of tissue integrity [1]. The down‐regulation of E‐cadherin in cells has been shown to be associated with the epithelial‐mesenchymal transition of tissues and apoptosis, leading to the assumption that E‐cadherin is critical to cellular survival and pluripotential [2]. On the basis of this assumption, two RNA interference (RNAi) transfection reagents, Lipofectamine ™ RNAiMax Reagent (Invitrogen®) and Accell™ siRNA Delivery (Thermo Scientific Dharmacon®), were used to decrease the gene expression of E‐cadherin in H9 stem cell lines to determine the signaling pathways of cellular differentiation. The results reveal that although E‐cadherin gene expression in H9 stem cells decreased after RNAi knockdown, the cells remained pluripotent. These results refute prior claims that the presence of E‐cadherin is crucial for cellular differentiation and imply that the down‐regulation of E‐cadherin is not associated with the epithelial‐mesenchymal transition of tissues and apoptosis. E‐cadherin may not be critical to cellular survival and pluripotential, providing a basis for more extensive research on signaling pathways of differentiation. Supported by NIH T34GM008411
Fyn kinase controls extracellular Ca2+ entry leading to degranulation in IgE‐antigen stimulated mast cells
Authors: Sánchez Elizabeth , Ibarra‐Sanchez Alfredo , Gonzalez‐Espinosa Claudia
Published: June 2021

Mast cells (MCs) express the high affinity IgE receptor (FcεRI) in their plasma membrane. This receptor binds monomeric IgE and, after antigen recognition, induces Ca2+‐dependent degranulation of mast cells. FcεRI receptor signal transduction system is regulated by two Src‐family kinases, Fyn and Lyn. Fyn kinase positively regulates degranulation but its role on the molecular mechanism that mobilizes intracellular Ca2+ has not been explored in detail. In this work, we analyzed by fluorometric techniques and pharmacological tools Ca2+ mobilization in bone marrow‐derived mast cells(BMMCs)obtained from WT and Fyn−/− mice stimulated by IgE‐antigen. We observed that Fyn−/− BMMCs showed a reduction in extracellular Ca2+ influx when stimulated with IgE‐antigen, compared to WT cells. The calcium channel blocker 2‐aminoethoxydiphenyl‐borane (2‐APB) was able inhibit maximal Ca2+ rise in WT but not in Fyn−/− cells. Ca2+ entry through 2‐APB sensitive channels was important for full degranulation, NFκB activation and mRNA accumulation of IL‐2 and CCL‐2 in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channel after IgE‐antigen stimulation, but it is not related to protein tyrosine phosphorylation. Our results strongly suggest that Fyn kinase activate TRPC calcium channels that contribute to receptor operated calcium entry in mast cells.
Cell‐Cell Contact Dependent Subcellular Localization of PAR‐3 Regulated by 14‐3‐3 epsilon
Authors: LIU TZUAN , LIOU JUN‐YANG
Published: June 2021

14‐3‐3 proteins function as adaptors that regulate multiple signal transduction pathways involved in the cell division, growth, and intracellular trafficking/targeting. Through binding to the phosphoserine motif of the polarity proteins, 14‐3‐3 proteins have been reported to play an important role in conferring cell adhesion and polarization, which are the fundamental processes in cell migration, division and development. In this present study, the roles of the 14‐3‐3ε (PAR‐5) and partitioning defective protein 3 (PAR‐3), a cell‐polarity protein required for establishing cell polarization and apicobasal asymmetries associated with cell adhesion, in the regulation of cancer cell adhesion and polarization was investigated in human hepatocellular carcinoma cells (HCC). Majority of PAR‐3 expression was determined to be localized at the plasma membrane, cytosol and cell‐cell contact interface by immunofluorescent confocal microscopy. The abundant expression of PAR‐3 was subsequently relocated and accumulated to cell‐cell contact while overexpressing 14‐3‐3ε, indicating the vital role of 14‐3‐3ε in regulating function and trafficking of PAR‐3. Moreover, the significant colocalization of PAR‐3 with 14‐3‐3ε and accumulation of catenins‐complex at the cell‐cell contact interface were observed in 14‐3‐3ε overexpressed cells. Molecular interaction of 14‐3‐3ε with PAR‐3 was further confirmed by analysis of protein immunoprecipitation. These results implied that 14‐3‐3ε involved in the modulation of cell polarization and adhesion which may potentially regulate cancer cell metastasis and proliferation.
The Role of β‐catenin in cell division and colon cancer
Authors: Alba Nancy , Anderson Darrell , Cargile TeAngelo , Rodriguez Giovanni , Romero Karla , Johnson Carol , Narayan Malathi , Twining Sally
Published: June 2021

Colon cancer is the fourth most lethal cancer in the U.S. As food passes through the colon, water and vitamins are absorbed and epithelial cells are sloughed off and replaced by a tightly regulated cell division process. Unregulated cell division can lead to the formation of polyps and tumors. β‐catenin plays a role in regulating cell division and was modeled by the Messmer SMART Team (Students Modeling A Research Topic) using 3D printing technology. In non‐dividing cells, a multi‐protein complex of APC, GSK‐3 and Axin phosphorylates β‐catenin, signaling its degradation and preventing cell division. When cell division is needed, a Wnt signal cascade causes the complex to release β‐catenin, stabilizing the protein for nuclear translocation and binding to TCF, a transcriptional activator, thus triggering cell division. Competitive binding of the inhibitor proteins, ICAT and Chibby, to β‐catenin negatively regulates this process. In colon cancer, mutations in APC or Axin impede binding of the complex to β‐catenin, preventing degradation, leading to increased nuclear localization, binding to TCF and deregulated cell division. Additionally, survivin, an anti‐apoptotic protein that enables tumor cell survival, is upregulated. Understanding β‐catenin's structure could help design drugs to promote binding of inhibitors to prevent the unregulated cell division of cancer. Supported by a grant from NIH‐NCRR‐SEPA.
Cellular Analysis Using the MILLIPLEX® MAP™ Akt/mTOR Signaling Pathway Panels
Authors: Hwang Joseph
Published: June 2021

The Akt/mTOR pathway plays a central role in a number of key cellular processes including translation, cell cycle, cellular energy homeostasis, and apoptosis. The study of this pathway is important for an understanding of signaling involved in physiological as well as several pathological states including cancer and diabetes. We have developed a Luminex®‐based multiplex assay that allows for the simultaneous detection of multiple phosphorylated Akt/mTOR pathway proteins in a single well. Using the Akt/mTOR Multiplex Panels we observed increased levels of phosphorylated IR, IGF1R, IRS1, Akt, GSK3α, GSK3β, p70S6K, and RPS6 in stimulated cells when compared to their unstimulated counterparts. Modifications in phosphorylation levels were confirmed by the treatment of cells with specific inhibitors, such as wortmannin which blocked phosphorylation of PRAS40, mTOR, TSC2, Akt, GSK3α, GSK3β, p70S6K, and RPS6, as well as with phosphatase treatment of cell lysates. The Akt/mTOR signaling panel results were more quantitative, sensitive, and required much less labor compared to Western blotting when used to analyze phosphorylated protein biomarkers in cellular lysates. Support for this research is sponsored by Millipore Corp.
Structural characterization of the interactions between CYP51 from Trypanosoma cruzi and Trypanosoma brucei and the antifungal drugs posaconazole and fluconazole
Authors: Chen Chiung‐Kuang Jack , Mckerrow James H , Podust Larissa M
Published: June 2021

Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14‐α‐demethylase (CYP51) which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti‐fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. We have now determined the crystal structures for CYP51 from T. cruzi co‐crystallized with the antifungal drugs fluconazole and posaconazole. The structures provide new insights into binding of azoles to CYP51 as well as mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti‐Chagasic drugs with improved efficacy and reduced toxicity.
Structural description of enzyme catalysing unusual modification in lantibiotic biosynthesis
Authors: Garg Neha , Velasquez Juan , Nair Satish , van der Donk Wilfred A
Published: June 2021

Lantibiotics are ribosomally synthesized antimicrobial peptides which undergo extensive post translational modifications. These post‐translational modifications confer biological activity on them. Epilancin 15 X was recently discovered and was found to be active against resistant strains of Staphylococcus aureus and Enterococci with MIC values <0.5μg/mL. Besides general modifications which include lanthionine and methyllanthionine rings, epilancin 15 X contains an unusual N‐terminal (R)‐2‐hydroxy propionyl group (Hop) which may provide it with its striking MIC values. Hop was speculated to be produced by dehydration of a serine residue in the precursor peptide. The leader peptide cleavage and hydrolysis of the resulting dehydroalanine residue would result in 2‐oxopropionyl group which could then be reduced to corresponding alcohol by a novel NADPH dependent oxidoreductase encoded by elxO within the biosynthetic gene cluster. ElxO is a 248 amino acid protein belonging to short chain dehydrogenase‐reductase (SDR) superfamily. It is a highly promiscuous enzyme which can be used to engineer and create novel lantibiotics. To gain further insights into the biosynthesis of this N‐terminal Hop residue, structural studies of ElxO were undertaken and structure was solved to a final resolution of 1.7 Å. Based on conserved residues in the structure, a catalytic mechanism was proposed.
Zoledronic acid increases anti‐angiogenic factors in an osteotropic breast cancer cell line
Authors: Morshedian Shaghayegh , Contreras Cynthia , Bush Jason A.
Published: June 2021

Bone is the most common organ for tumor metastasis in breast cancer patients. Currently, bisphosphonates are a main‐stay of cancer treatment known to reduce and/or delay the skeletal related events of malignancy by impairing osteoclast‐mediated bone resorption. The purpose of this project was to define differences between the breast cancer cell line, MDA‐MB‐231, and an osteotropic clone, MDA‐231BO, in response to the bisphosphonate, zoledronic acid (ZOL). Results demonstrate that MDA‐231BO cells are more sensitive to ZOL cytotoxicity and the salutary effects are both media‐ and calcium‐dependent, as elevating exogenous calcium levels increased ZOL cytotoxicity. To investigate possible mechanisms of ZOL‐induced cell death, total RNA, whole protein lysates, or mitochondrial‐enriched fractions were isolated from treated MDA‐231BO cells and compared to non‐treated controls. Using semi‐quantitative RT‐PCR, ZOL‐treated cells exhibited a down‐regulation of CYR61, a matricellular protein over‐expressed in highly invasive cancers while using a combination of 2D‐PAGE separation and MALDI‐MS/MS, we report the identification of a small subset of differentially‐expressed proteins including an up‐regulation of anti‐angiogenic proteins. Taken together, our results provide a better understanding of the molecular mechanism associated with ZOL exposure in osteotropic cancer cells.
Defending Against Infection: Quorum Quenching by Enzymes in Human Liver Microsomes
Authors: Davis Amy Miller , Haines Donovan C.
Published: June 2021

Some bacterial pathogens like rely on a process called quorum sensing to control gene regulation. A medically important example is Pseudomonas aeruginosa, a pathogen that is a frequent cause of death for cystic fibrosis patients. In recent years hosts, including humans, have been shown to interfere with bacterial quorum signaling by enzymatically destroying the small molecule signals themselves, a process termed quorum quenching. Investigations into the inner workings of quorum quenching enzymes led us to assess the ability of human liver microsome P450 cytochromes (P450s, Phase I drug metabolizing enzymes) to inhibit or limit acyl homoserine lactone (AHL) quorum sensing as detected in a A.tumefaciens AHL bioassay. Results indicated that P450s can inhibit quorum sensing activity up to 80%. Additionally, studies were performed to assess the potential of UDP‐glucuronic acid transferases (UGTs, Phase II drug metabolizing enzymes) to further inactivate the AHL signals. Results showed UDP‐glucuronic acid dependent quenching similar to the NADPH‐dependent quenching obtained with P450s. UGTs quenched AHL quorum activity 50–70%, slightly less active than the P450s. Assays indicated that UGTs and P450s maintain their quenching ability in combination, with slightly higher quenching activity than either enzyme alone. While studies continue, these results are a good indication that P450s and UGTs play a role in defending us against infection by AHL‐dependent pathogens. These studies have major implications for infection in general and specifically in cystic fibrosis, where some P450s are strongly down regulated and this may contribute to Pseudomonas aeruginosa sensitivity.
Targeting both “Half‐Reactions” of a Glycokinase Enzyme for Drug Discovery
Authors: Olson Matthew W. , Lanter James , Sun Weimei , Struble Geoff , Gaul Michael , Lawrence Kuo , Todd Matthew
Published: June 2021

With regard to drug discovery, two glycokinases are of particular interest based upon clinical data; ketohexokinase (EC 2.7.1.3, 1‐3) and glucokinase/hexokinase 4 (EC 2.7.1.2, 4‐8). Yet, modulators for either target have had limited success in pharmaceutical development. Despite, decades of basic and applied research, only a few small molecule activators for glucokinase have made it to phase 1 clinical trial. This could in fact, be due to the basic assay designs used for and the initial steps in drug discovery. Traditionally, glycokinase activities have been monitored by radioactive, enzyme‐linked, fluorescence polarization detection of ATP turnover. Described herein is a novel label‐free means of directly monitoring the bi‐bi reaction for a ketohexokinase where detection of enzyme activity utilizes mass spectroscopy. Here, turnover of ATP and fructose to ADP and fructose 1‐P, respectively, can be monitored directly and simultaneously. The selectivity of analyte detection is maximized by MRM, allowing for detection of each analyte by LCMSMS from both cells and tissues. Basic enzyme characterization is demonstrated using HTMSMS. In addition, the mechanisms of action for two unrelated compounds and the implications of target engagement of pharmaceutically active compounds from “test tube” to tissue samples are discussed.
Structural studies in the biosynthesis of the glycopeptide antibiotic teicoplanin
Authors: Li Zhi , Nair Satish K.
Published: June 2021

Teicoplanin is a glycopeptide antibiotic that has proven efficacy against Gram‐positive bacteria and is in clinical trials. It is distinguished from vancomycin‐type glycopeptides antibiotics by side chain modifications, which have been shown to contribute to its improved efficacy and superior pharmacokinetic profile. Our research aims to study the biosynthetic mechanisms of teicoplanin, which will likely lead to the development of new glycopeptide derivatives. Here we present high resolution crystal structures of several enzymes that modify teicoplanin side chains during its biosynthesis. The structures provide insights into the active residues of these enzymes and suggest possible mechanisms of modifying glycopeptides antibiotics.
An anti‐inflammatory peptide combination reduces oxidative effect of plasma from cardiomyopathy patients
Authors: Elboudwarej Omeed , Kim J. Brian , Hama Susan , Hough Greg , Fonarow Gregg
Published: June 2021

Background

Patients with cardiomyopathy are under increased oxidative pressure and their plasma shows a high plasma inflammatory index (PII). In the present study, our group tested whether the combination of the anti‐inflammatory peptides apoJ[113–122] and FREL would effectively reduce the PII.

Methods

Plasma from 10 patients with heart failure was incubated with 0.5 μg/ml apoJ[113–122], with 2 μg/ml FREL, or with a combination of the two peptides. The plasma was then diluted 2000‐fold and incubated with human endothelial cells. To determine the PII, we used a monocyte chemotactic assay (MCA) to quantify the plasma‐induced inflammation of the endothelial cells.

Results

Treatment of plasma with a combination of peptides has an additive effect on improving the PII. The PII value was reduced by 80% with apoJ[113–122] alone, 52% with FREL alone, and 127% in combination.

Conclusion

Treatment of plasma with a combination of peptides resulted in additional improvement of the PII. One potential explanation is that these peptides have an additive effect in sequestering or preventing the formation of different lipid oxidation products, possibly by forming biochemical complexes that are more active than the peptides are individually. Grant source: U.S. Public Health Service Grants HL‐30568 and HL‐34343 and the Laubisch, Castera, and M.K. Gray Funds at the University of California Los Angeles.
Anti‐Glycation Effect of Gold Nanoparticles
Authors: KIM JI HOON , HONG CHUNG‐OUI , NAM MI‐HYUN , LEE HYUN AH , YANG SUNG‐YOUNG , LEE JI‐YOUNG , LEE KWANG‐WON
Published: June 2021

In this study, we investigated anti‐glycation effects on gold nanoparticles. It has been known that protein aggregation leads to make advanced glycation end‐products (AGEs) synthesis, and AGEs can be a diabetes‐ and aging‐causing factor. The aqueous solution (500mL) of 1–2 mM HAuCl4 was brought to a reflux and stirred, and then 50mL of 38.8mM trisodium citrate solution was added quickly, which resulted in a solution color change from pale yellow to deep red. We employed SEM to measure their sizes of gold nanoparticles, and glycation reaction with BSA and glycoaldehyde. Aminoguanidine, a well‐known anti‐glycation compound, is used as a positive control. The reaction mixture contained 6.02*10^17 atoms/mL and 1.204*10^18 atoms/mL of Au. We found that 13 nm‐nano sized gold particles showed anti‐glycation effects dose‐dependently suggesting a possible role of these particles against protein glycation reaction.
Metagenomics ‐ the next structural frontier
Authors: Joachimiak Andrzej
Published: June 2021

Metagenomics aims to analyze, reconstruct and characterize how microbial communities organize and evolve their collective genomes to thrive in the environments they co‐habit. Human microbiomes harbor a large number of microbial species, many of which are uncharacterized or unculturable. The human gut microbiome represents a highly complex microbial community that has a significant impact on human physiology. This microbiome is believed to significantly enhance the metabolism of amino acids, carbohydrates, xenobiotics, methanogenesis, biosynthesis of vitamins and other compounds. Therefore, microbiomes represent a potential pool of genes coding for novel proteins and functions. The Protein Structure Initiative (PSI) human gut microbiome pilot project has been initiated with emphasis on the guts of healthy and diseased individuals. The PSI is well positioned to study proteins from microbiomes, and its high throughput protein production and structure determination pipelines are well suited to the microbial proteins that emanate from these projects. We have analyzed gut metagenomic data to identify novel, previously uncharacterized, human microbiome‐specific protein families. A set of reagent genomes was used to extract genes from gut microbiome and a number of structures from normal and enteric microflora have been determined. The structural and functional results will be discussed. This work was supported by National Institutes of Health Grant GM074942 and by the U.S. Department of Energy, OBER, under contract DE‐AC02‐06CH11357.
NIGMS PSI:Biology Initiative ‐ Enabling High‐Throughput Structural Biology and Structural Genomics
Authors: Preusch Peter C. , Basavappa Ravi , Chin Jean , Edmonds Charles , Flicker Paula , Lewis Catherine , Smith Ward , Wehrle Janna , Berg Jeremy
Published: June 2021

The new Protein Structure Initiative (PSI:Biology) to be funded by NIGMS beginning July, 2010 will apply high‐throughput structural methods to important biological problems. This will be accomplished by establishing partnerships between centers for structure determination and biologists with interests in particular groups of proteins. The PSI:Biology network centers will include: 1) Centers for High‐Throughput Structure Determination, 2) Centers for Membrane Protein Structure Determination, 3) the PSI:Material Repository, and 4) the PSI:Biology Knowledgebase. Consortia for High‐Throughput‐Enabled Structural Biology Partnership awards will define targets for structure determination, support functional studies in the partners’ laboratories, and support part of the cost for structure determinations by the centers. Researchers may also suggest specific proteins for structure determination through the PSI Community Nomination site. In addition to protein structures and models, the network will generate and make available reagents and plasmids for expressed proteins to support functional studies by the research community at large. Capabilities of the centers, examples of current and potential future partnerships, and opportunities to apply for PSI:Biology grant support will be presented. For details see: http://www.nigms.nih.gov/Initiatives/PSI/psi_biology/.
Combining High‐Throughput Structure Determination and Enzyme Characterization for Functional Annotation of a Large Enzyme Superfamily
Authors: Burley Stephen Kevin
Published: June 2021

Under the auspices of the NIH Protein Structure Initiative (PSI), the New York SGX Research Center for Structural Genomics (NYSGXRC) developed a high‐throughput X‐ray crystallography platform that currently produces more than 200 de novo protein structures annually at an average cost of ~$40,000 per structure. As required by the PSI, ~15% of NYSGXRC efforts have been devoted to studies of targets nominated by members of the scientific community. The majority of our Community Targets were pursued in collaboration with an NIH‐funded P01 Program Project, entitled “Deciphering Enzyme Specificity” (GM071790; Principal Investigator: John Gerlt). Together, the P01 and the NYSGXRC examined the large Enolase/Amidohydrolase enzyme superfamily, with the goal of establishing a scalable experimental/bioinformatic platform for large‐scale functional characterization that supports prediction of enzyme function from sequence or structure. To date, the NYSGXRC has determined experimental structures of more than 70 Enolases and 30 Amidohydrolases plus more than 20 operon/pathway‐related enzymes. Illustrative successes from this synergistic systems structural genomics collaboration will be presented.
Structural Genomics – When are Enough Structures Enough?
Authors: Levitt Michael
Published: June 2021

Deciding when protein structures have been solved in sufficient numbers to empower all of biology with structural information is difficult as it depends on so many factors that are scientific, statistical, political and sociological. Scientists need to estimate how big is the universe of all proteins sequences and also how function comes about from sequences and their combinations to decide if this the protein universe is indeed finite. Statisticians need to estimate the rate of progress being made by current levels of effort to estimate the likely dates and costs of any desired level of structural coverage. Politicians need to weigh the costs of these efforts against the costs of competing projects. Finally, social factor such as the interest of experimental biologist in protein structure need to be considered so as be able to maximize the use of the information. I will dare to dip into these murky waters but cannot promise a magic solution.