bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of the Anti‐inflammatory Action of Pulsatile Laminar Flow: Role of AMPK in Chromatin Remodeling
Authors: Flores Leona , Li Yi-Shuan , Chien Shu
Published: June 2021

Pulsatile laminar flow (PS) mediates many anti‐inflammatory functions in endothelial cells (ECs). However, the mechanisms involved in the repression of inflammatory responses by mechanical stimuli have not been elucidated. Transcriptional repression can occur through epigenetic modifications of gene promoter regions. We performed experiments to test the hypothesis that the PS‐dependent protective mechanism occurs through signaling pathways that downregulate the expression of inflammatory and adhesion molecule genes and that this mechanotransduction process is mediated through an AMP‐activated protein kinase (AMPK) pathway and subsequent chromatin modifications. We found that 24‐hr PS downregulated EC gene expression of monocyte chemoattractant protein‐1 (MCP1) and vascular cell adhesion molecule‐1 (VCAM) and inhibited monocyte attachment to ECs via an AMPK‐dependent pathway. PS also induced the AMPK‐dependent recruitment of the corepressor enzymes histone deacetylase‐3 and ‐5 to the MCP1 and VCAM proximal promoters as well as concomitant histone hypoacetylation. Our findings indicate that PS suppresses inflammatory processes through chromatin modifications and downregulation of AMPK‐dependent inflammatory genes and monocyte adhesion, thus providing new insights into the role of chromatin remodeling in mechanotransduction. 3R01 HL106579 (S.C.) and 3R01 HL106579‐01S1 (L.F.)
Obscurins Invade Breast Cancer Research
Authors: Shriver Marey , Kontrogianni-Konstantopoulos Aikaterini
Published: June 2021

Obscurins are giant multidomain proteins, encoded by the single OBSCN gene, which play important roles in the structural organization and contractile activity of striated muscles. Recently, genetic linkage analysis identified OBSCN and TP53 as the only common and most frequently mutated genes in breast and colorectal cancers among 13,023 genes studied. Our laboratory set forth to study the expression profile of the OBSCN gene in breast cancer. Our data revealed that obscurins are readily expressed in normal breast epithelial cells (MCF10A), but their amounts are significantly diminished in breast cancer cells (MCF7 & MDA‐MB‐ 231). Down‐regulation of obscurins in MCF10A cells via siRNA technology resulted in decreased levels of β‐catenin and Ecadherin, both of which are major components of adherens junctions (AJ) and are heavily implicated in the formation and metastasis of breast tumors. Residual β‐catenin showed a preferential nuclear accumulation in obscurin‐null MCF10A cells, and a concomitant loss from cell‐cell junctions. Interestingly, TGF‐β1 treatment of normal MCF10A cells resulted in down‐regulation of obscurins, β‐catenin, and E‐cadherin. In summary, our findings indicate that TGF‐β1 may negatively regulate the expression of obscurins in breast epithelium, loss of which results in diminished levels and altered distribution of major AJ proteins, and thus disruption of cell‐cell contacts.
Lack of N‐Sulfation of Podocyte Cell Surface Heparan Sulfate Glycosaminoglycans Leads to Abnormalities in Podocyte Organization, Adhesion, and Migration
Authors: Sugar Terrel A , McCarthy Deborah J. , Esko Jeffrey D. , Holzman Lawrence B. , McCarthy Kevin J.
Published: June 2021

Previous work showed that cell surface heparan sulfate (HS) glycosaminoglycans (GAG) modulate podocyte (podo) organization on the glomerular basement membrane (Kid. Int. 74: 289–299). This study examined the role of HSGAG sulfation in this process by podo‐specific deletion of NDST1, the enzyme responsible for N‐sulfation of HSGAG. A novel mutant mouse (2.5P‐Cre/NDST1fl/fl) and immortalized podo line (NDST1‐null) were developed using a Cre‐lox approach. The kidneys in mutant mice appeared normal compared to controls, the only measurable difference in routine LM studies was a slight glomerular hypertrophy in the mutant mice. Electron micrographs from mutant animals showed mild podo foot process effacement and abnormal podo attachment to Bowman's capsule, suggesting that podo‐matrix interactions were affected. NDST1‐null podo had a significantly decreased ability to adhere, spread, and migrate upon fibronectin compared to controls. Mutant podo also failed to assemble focal adhesions as efficiently as control cells. These results indicate that HSGAG sulfation is a critical mediator of cell surface proteoglycan (PG)‐matrix interaction, highlighting the importance of HSPGs in orchestrating podo cell behavior.

This work is supported by: NIDDK RO1‐DK077860

Grant Funding Source: NDDK RO1‐DK077860
MMP expression by intervertebral disc cells is responsive to changes in extracellular osmolarity
Authors: Cui Ying , Lee Sheena , Yu Jing , Urban Jill
Published: June 2021

We examined the response of cells from the central regions of the intervertebral disc, the nucleus pulposus (NP) to changes in extracellular osmolarity with particular reference to matrix metalloproteinases (MMPs) expression.

NP cells were extracted from adult bovine caudal discs by enzyme digestion and cultured in alginate beads to maintain phenotype. MMP production was investigated by zymography and Western blotting, and total RNA was extracted from the cells and gene expression analysed using the Affymetrix GeneChip Bovine Genome Array.

MMP‐2, MMP‐3 and MMP‐13 were significantly upregulated when cells were cultured in hypo‐osmotic medium i.e. 260 mOsm seen in severely degenerate discs compared to 356 and 489 mOsm seen in healthy discs. A similar upregulation was seen at the gene level.

Our results show that expression of MMPs by disc cells is regulated by extracellular osmolarity, with MMP expression rising as osmolarity falls. These results could explain in part the findings that MMP expression increases with degree of disc degeneration i.e. with loss of aggrecan and fall in extracellular osmolarity.

Grant Funding Source : AAA
Sex Differences and Variation in Lower Limb Element Lengths: Implications for Physical Therapeutic Treatment of Anisomelia
Authors: Weir Alicia Suzanne , Márquez Samuel
Published: June 2021

Lower limb length discrepancy (LLLD) is a common condition affecting large portions of the general population and has been related to many biomechanical abnormalities in posture, pathological stress on joints, and modification of locomotor gait. Structural limb length discrepancy involves asymmetry in relative lengths of individual lower limb elements. Our study aimed to eliminate the error associated with using surface anatomical landmarks to measure limb length by instead examining the actual bony anatomy of the lower limb via disarticulated lower limbs from donor bodies at the SUNY Downstate School of Medicine. Limb length was calculated as the combined lengths of the femur & tibia. A total of 41 specimens were used (15 males & 26 females). Results showed that there were no significant (p<0.05) sexual differences in femur or tibia asymmetry. Femur length exhibited greater relative asymmetry than tibia length but this difference was not significant (p<0.05). The mean difference between left and right femur length was 48.37 millimeters (mm) while left and right mean tibia length differed by 49.6 mm. Total left limb length was, on average, longer than on the right by 1.78 mm. LLLD patients may require rehabilitative therapeutic protocols that are patient‐specific rather than general since this condition may be attributed to differences in overall limb length rather than femur or tibia length alone.

Grant Funding Source: Alumni Association of SUNY Downstate College of Medicine
Functionalized Nanocoating for Accelerated Wound Healing
Authors: Kulkarni Rutuja Prakash , Mills David Keith
Published: June 2021

Multilayered nanofilms, functionalized with growth factors, were developed to act as a reparative scaffold, stimulating cell proliferation, differentiation and tissue formation in a skin wound model. Poly L‐Lysine and Poly L‐Glutamic Acid, incorporating platelet derived growth factor and transforming growth factor‐β, were electrosprayed onto a wounded skin equivalentas a ten‐bilayer nanocoating and cultured for a 6‐day period. Control cultures received an applied nanocoating with no growth factors. Zeta potential (ZP) & quartz crystal microbalance (QCM) data were used to characterize film construction. Histochemical (HC) and immunohistochemistry (IHC) analyses were performed to assess cell proliferation and tissue formation. ZP and QCM data indicated that the nanocoatings were deposited evenly & maintained a charge differential between layers. HC and IHC results show that proliferation was highest in epidermal vs. dermal layers and IHC studies suggest that histogenesis was greater in the dermis. Control cultures showed a very poor wound response. The quality of tissue regenerated will determine the large‐scale scope of this therapy but preliminary results suggest a potential new treatment modality. Funding provided by the State of Louisiana Governor's Biotechnology Initiative.
The role of Brahma‐related gene 1 in regulating the expression of microRNAs in colonic smooth muscle cells
Authors: Chen Meng , Herring Paul
Published: June 2021

To identify microRNAs (miRs) that are regulated by Brahmarelated gene 1 (Brg1) in smooth muscle cells (SMCs), we performed microRNA array screens of colonic smooth muscle. Quantitative reverse transcription‐polymerase chain reaction assays confirmed that 6 miRs were down‐regulated and 6 up‐regulated in smooth muscle specific Brg1 knockout tissue. Inactivation of endogenous Brg1 by introducing dominant negative Brg1 into wild type SMCs in vitro, revealed that miR‐143/145 are likely to be directly regulated by Brg1 in smooth muscle cells. miR‐143/145 have been previously shown to be regulated by myocardin and to be important for regulating the phenotype of smooth muscle cells. In Brg1 null SW13 cells, miR‐143/145 were dramatically induced by myocardin only in the presence of Brg1. Chromatin immunoprecipitation assays demonstrated that myocardin increased the binding of serum response factor to the miR‐143/145 promoter only in the presence of Brg1. Together these data suggest a mechanism in which Brg1 containing SWI/SNF complexes are required for myocardin to induce expression of miR‐143/145 in smooth muscle. This suggests that SWI/SNF mediated chromatin remodeling regulates the phenotype of smooth muscle not only by directly affecting expression of smooth muscle specific genes, but also by regulating expression of microRNAs that further modulate expression of their targets.

Supported by NIH DK61130.
Transcription factor access to their sites in chromatin
Authors: Floer Monique , Gjidoda Alison
Published: June 2021

We have recently discovered a novel mechanism for exposing transcription factor binding sites in chromatin in the yeast S. cerevisiae (Floer, M. et al., 2010 Cell, 141, 407). The mechanism involves trapping of a nucleosome at a specific position and partial unwrapping of the nucleosomal DNA ends, which leads to the incorporation of a segment of DNA around 30 bp shorter than that associated with an ordinary nucleosome. At the UASg, the regulatory region in the GAL locus of yeast, such a structure is created by the so‐called nucleosome remodeler RSC, which recognizes specific sites in the UASg. Formation of the structure facilitates binding of the activator Gal4 and expression of the associated genes. Nucleosomes that are part of structures such as the one at the UASg have been missed in previous studies due to an assumed size of 147 bp for a DNA fragment associated with a nucleosome. But we find that many similar structures are found in the yeast genome, and are often associated with sites for transcription factor binding. This suggests that partial unwrapping of a nucleosome may be a common mechanism for exposing transcription factor binding sites in yeast. Using the methods we have developed we are now analyzing how transcription factors of higher eukaryotes access their sites in chromatin, taking the pro‐inflammatory genes of mouse macrophages as a paradigm for gene induction in response to environmental stimuli. The goal of these studies is to understand how chromatin contributes to lineage specific activation and repression of genes.
A Dominant Role for an Error‐Prone DNA Polymerase in Recombination Drives Evolution
Authors: Pomerantz Richard T , Kurth Isabel , Goodman Myron , OˈDonnell Mike
Published: June 2021

Homologous recombination (HR) repairs double‐strand breaks in DNA by directing replication to copy sequence information from a homologous donor DNA. Importantly, this mode of repair is considered error‐free which implies that only high‐fidelity DNA polymerases (pols) perform recombination‐directed replication (RDR). Yet in E. coli, error‐prone Y‐family translesion pols function with RecA recombinase, are highly upregulated in response to stress, and promote mutations following DSBs, which suggests a possible role for these mutagenic pols in HR. Here, we investigate E. coli translesion pol activity in HR by reconstituting RDR in vitro. We find that translesion pol IV is proficient in RDR even without the β‐processivity clamp, whereas translesion pol V lacks this activity. Notably, low‐fidelity pol IV initiates RDR more efficiently than high‐fidelity replicative pol III and is preferentially recruited to D‐loop recombination intermediates under stress response conditions. Unexpectedly, we find that pol III is inhibited in RDR due to its proofreading activity which is stimulated at D‐loops. Finally, we show that pol IV promotes mutations during RDR. These data demonstrate a dominant role for error‐prone pol IV in HR and elucidate the molecular basis of adaptive mutagenesis which facilitates evolution under conditions of stress.
NIP45 Promotes Telomere Targeting to PML Bodies in ALT Cells
Authors: Farley Demetra D , Yu Hongtao
Published: June 2021

Telomere length maintenance is critical for continued cell proliferation. Certain cancer cells do not have active telomerase and use homologous recombination to lengthen their telomeres, a mechanism referred to as alternative lengthening of telomeres (ALT). We have previously shown that the SMC5/6 complex is required for telomere length maintenance in ALT cells. The SMC5/6 component MMS21 sumoylates multiple telomere‐binding proteins and promotes telomere targeting to ALT‐associated PML bodies (APBs). In this study, we show that the SUMO‐like protein NIP45 is also required for targeting telomeres to APBs. NIP45 itself localizes to PML bodies and promotes MMS21‐dependent sumoylation of telomere‐binding proteins. The C‐terminal SUMO‐like domain of NIP45 is sufficient to restore both APB formation and sumoylation of telomere‐binding proteins in NIP45 RNAi cells. Our results suggest that NIP45 enables PML‐body recruitment and sumoylation of telomere‐binding proteins, thus promoting APB formation in ALT cells.

This work was supported by a National Research Service Award grant 5 T32 GM 8203‐24 to Demetra D. Farley.
The isoprenoid perillyl alcohol inhibits telomerase activity in prostate cancer cells
Authors: Sundin Tabetha R , Gauthier David , Peffley Dennis , Hentosh Patricia
Published: June 2021

Plant‐derived isoprenoids suppress carcinogenic processes in vivo and in vitro. Previously we established that the isoprenoid, perillyl alcohol (POH), acted mechanistically on translation of specific proteins through modulation of mammalian target of rapamycin (mTOR) signaling. Telomerase–the enzyme responsible for immortalizing cells through addition of telomeric repeats–is derepressed early in an aspiring cancer cell. We examined effects of biologically‐relevant concentrations and short incubations (1–16 h) of POH or the mTOR inhibitor, rapamycin, on telomerase activity in prostate cancer cell lines. A rapid suppression of telomerase activity (from 70 to >95%) was observed by real‐time quantitative telomerase repeat amplication protocol and confirmed by polyacrylamide gel analysis. However, human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered as determined by quantitative RT‐PCR. Western blot analysis revealed that hTERT protein levels decreased in response to POH or rapamycin, but E3 ligase Makorin‐1–which is thought to signal proteolytic breakdown of hTERT–was not detectable. No change in hTERT phosphorylation at Ser824 was observed, and no cellular redistribution of hTERT protein was found. These findings provide evidence for a unique link between nutrient‐ and macrolide‐mediated regulation of mTOR and hTERT‐a key factor regulating DNA structure and stability.
Development of Bovine Immunodeficiency Virus (BIV) Tat Peptide Analogs as Inhibitors of BIV Transcription
Authors: Grist Jonathan J , Wright Heather J , Collins Elaine D
Published: June 2021

Bovine immunodeficiency virus (BIV) is an advantageous model system for studying human immunodeficiency virus (HIV) due to its genetic similarity, structure, and mechanism of infection. The key to pathogenicity is the interaction between the trans‐activating (Tat) peptide and the transactivator response element (TAR) RNA in both human and bovine infections. The interaction of the Tat peptide and TAR RNA during the HIV life cycle causes viral RNA to fully transcribe more efficiently and accumulate in the nucleus of its host. Methods of inhibition of HIV have been diligently studied over the last 30 years with the best achievements found in combination therapy using small molecule drugs. Recently, pharmaceutical industries have identified the benefits of protein therapeutics and this field has been growing rapidly. We are interested in the use of such biological inhibitors against HIV, such as analogs of the Tat peptide, to competitively bind TAR RNA and halt viral pathogenesis. We have synthesized wild‐type, two single mutants (R78G & K75G), and one double mutant (R78G/K75G) Tat peptides, using molecular techniques. These peptides have been characterized using UV and mass spectrometry. The TAR cDNA has been successfully constructed and reverse transcribed into homogeneous TAR RNA. The TAR RNA has been characterized using UV spectrometry. We will test the binding affinities of each peptide to TAR RNA using isothermal titration calorimetry (ITC) in hopes of finding stronger binding of at least one of the mutants. Our goal is to develop therapeutic agents that interfere with the interaction between TAR RNA and Tat peptides. The results from this research can be applied to the synthesis and use of HIV Tat peptide analogs in hindrance of the viral infection in humans. We would like to thank the National Institute of Health grants 5T34GM008253 and 5R25GM071381 for funding this project.
tRNA wobble‐uridine modification pathways play critical roles in maintaining growth under nutrient limitation by altering the translational capacity of the cell
Authors: Laxman Sunil , Tu Benjamin P
Published: June 2021

Several eukaryotic tRNAs have modified nucleosides at the anticodon site. The uridine at the first anticodon position of Lys, Glu or Gln tRNAs is methylated at position 5 and/or thiolated at position 2 by proteins putatively regulated by the TORC1 pathway. These modifications are thought to alter tRNA anticodon‐codon interactions, improving decoding during translation. However the biological importance of these modifications for cell growth or survival, or the relative roles of uridine thiolation vs methylation is unknown. In this study, using prototrophic strains of S. cerevisiae, we show that tRNA uridine thiolation pathways are essential for yeast to maintain metabolic cycles and synchronous cell division under carbon limitation. We also suggest differences in the relative importance of uridine thiolation and methylation pathways. Under nutrient limitation, cells lacking these pathways show reduced translation of specific transcripts. Finally, we show that cells alter relative levels of tRNA wobble‐uridine methylation vs thiolation in response to the availability of specific amino acids. We suggest that these tRNA modification pathways play opposing roles in increasing translation/growth and quiescence/survival, with cells fine tuning translation in response to nutrients by altering levels of these modifications. Research support: grants NIH R01GM094314 and CPRIT RP110223 to BPT.
Use of 2‐Aminopurine to detect positional ambiguity in group II single‐ nucleotide bulge loops
Authors: McMichael Elizabeth L. , Serra Martin J.
Published: June 2021

Six RNA hairpin sequences containing a single‐nucleotide bulge loop with the fluorescent analog 2‐aminopurine (2‐AP) were optically melted in 1M NaCl and subject to fluorescent spectroscopy. Four of the bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity in the exact position of the bulge. The structural similarity to adenine and the fluorescent nature and properties of 2‐AP were used to analyze the uncertainty in position by inserting the fluorophore into the bulge loop on the 5′ and 3′ sides of the hairpin stem. The two remaining hairpins act as controls, with one containing a group I bulge loop and the other being fully paired. The group I bulge loop, which contains an unpaired nucleotide, showed a decrease in fluorescence as temperature increased, where the fully paired hairpin showed an increase in fluorescence with temperature. Previously, structural probing of group II bulge loops within hairpin motifs (McCann et al. 2011) indicated that the bulged nucleotide is the one positioned farther from the hairpin loop. This study suggests that when 2‐AP exists in group II bulge loops as the nucleotide closer to the hairpin loop, it exhibits behavior indicative of an unpaired nucleotide.
Investigating the Role of the Nematode‐Specific FAR Family of Proteins
Authors: Neutzling Kari Ann , Foudjet Armand Cedric , Comella Phillip , Schlotterback Mark , Bath Jennifer
Published: June 2021

Nematode genomes encode for proteins specific to the phylum Nematoda, which bear crucial importance for understanding nematode biology. Proteins specific to the phylum Nematoda are ideal targets for drugs with low toxicity to the host and the environment. Despite the importance of nematode‐specific proteins and protein families, few have been identified and very little information regarding them is available on public databases. Our work involves a novel, nematode‐specific family of proteins, the fatty acid and retinol binding (FAR) proteins of the phylum Nematoda. It was recently discovered that parasitic nematodes of plants, animals and humans secrete these structurally novel FAR proteins into the tissues they occupy. This family of proteins has been shown to be highly conserved across both plant and animal parasitic nematodes, yet there is limited information available regarding this family of proteins and their in vivo function(s) are poorly understood. Our specific research goals are to evaluate the ligand binding properties of FAR protein mutants created via multi site‐directed mutagenesis (MSDM). Constructs that do not bind the most commonly screened ligands for FAR proteins will be used to ascertain data on sequence variation and amino acids imperative for ligand binding. To date, we have created approximately 50 mutants containing two or more mutations. We expect that our research on this family of proteins may be significant for a broad range of scientists and agriculturalists in understanding plant and animal parasitism, nematode evolution and parasite‐control methods.
Development of RNAi tools for studying Leishmania (Viannia) and their application to the study of the Leishmania flagellum
Authors: Lye Lon-Fye , Owens Katherine , Notton Timothy , Acino Erin , Marcus Joseph , Beverley Stephen M.
Published: June 2021

RNA interference (RNAi) is a powerful tool for genetic analysis. While many Leishmania species lost the RNAi pathway deep in evolution, species within the Leishmania subgenus Viannia retain RNAi pathway genes. Correspondingly, introduction of constructs expressing dsRNA in L. (V.) braziliensis resulted in substantial down regulation of all target mRNAs tested. We are optimizing and developing protocols that will facilitate the use of RNAi and applied them to the study of the Leishmania flagellum. We developed vectors allowing rapid generation of stem‐loop (StL) constructs using Gateway (Invitrogen®) technology, and are working on methods for inducible RNAi and maximization of RNAi activity. The Leishmania promastigote flagellum contains paraflagellar rod (PFR); RNAi of genes encoding two major proteins PFR1 or PFR2 yielded parasites lacking the PFR and exhibiting motility defects. We probed the role of the intraflagellar transport pathway (IFT) by targeting four genes implicated in anterograde or retrograde trafficking. We were unable to recover transfectants bearing the respective StL constructs. The same StL constructs were transfected into an Argonaute null mutant which lacks a functional RNAi pathway. All IFT StL constructs were now successfully introduced into the Argonaute mutant. These data suggest that the IFT pathway is essential for the viability of L. braziliensis promastigotes.
Involvement of the Adenine/Uridine‐Rich Element Binding Proteins and microRNA Pathway in the Regulation of Human Interleukin‐3 mRNA
Authors: Martinez Marina , Marin Paula , Gonzalez Jose A. , Gonzalez Carlos I.
Published: June 2021

Human Interleukin‐3 (hIL‐3) is a cytokine that promotes myelopoiesis, differentiation of macrophages and granulocytes. Aberrant expression of this pleiotrophic factor has been involved in multiple myeloma and other hematological cancer. hIL‐3 harbors Adenine/Uridine‐Rich Elements (AREs) in its 3′‐UTR that mediate its post‐transcriptional control. The main objective of this study is to characterize the role of the ARE‐mediated regulatory mechanism and the microRNA‐mediated pathway in the post‐transcriptional regulation of the hIL‐3 transcript. Previous results from our laboratory identified five ARE‐BP complexes from approximately 30 to 90 kDa using UV crosslinking assays. Gel retardation experiments identified HuR, an extensively studied ARE‐BP, as a potential factor that recognizes the hIL‐3 ARE and may mediate the post‐transcriptional regulation of hIL‐3 mRNA. Also, a RNA affinity approach coupled with Western blot analysis and RT‐PCR confirmed the presence of HuR as a component of this ARE‐BP complex. More recent studies in the laboratory have suggested that the hIL‐3 3′‐UTR, which mediates its interaction with HuR, is required to enhance IL‐3 protein levels during T‐cell activation. Bioinformatics analysis using TargetScan suggests that miR‐15a and miR‐16 recognize sequences within the hIL‐3 3′‐UTR. Together, these observations imply that both the AREmediated post‐transcriptional mechanism and the microRNA pathway play important roles in the regulation of hIL‐3 during T‐cell activation. Ultimately, elucidating the role of these post‐transcriptional pathways in hIL‐3 expression can provide powerful therapeutic targets for the development of effective drugs against multiple myeloma and leukemias caused by abnormal hIL‐3 expression.
Dissecting the interactions between Human Argonaute proteins and RISC components
Authors: Chen Yen-Chun , Huang Jin-Wen , Chu Chia-Ying
Published: June 2021

MicroRNAs (miRNAs) are a group of non‐coding RNAs, with ~20–24 nt in length, that regulate gene expression at post‐transcriptional level. In human cells, miRNAs are loaded to Argonaute proteins (AGOs), which interact with other proteins to form RNA‐induced silencing complex (RISC). miRNAs then guide RISC to recognize their target mRNAs. These miRNA‐RNA‐protein complexes will trans‐locate to a specific cytoplasmic compartment called RNA processing body (P‐body), for the mRNA storage or degradation. It has been revealed that RCK/p54 (DDX6) is the key protein for P‐body formation and it also interacts with AGO2. Nevertheless, how RCK/p54 interacts with AGO2 to facilitate the miRNA‐mediate gene regulation in P‐body remains unclear. Here we report the domains and residues of AGO2 required for interacting with RCK/p54. Our preliminary data shows that the localization of AGO2 at various cytoplasmic RNA granules and the interaction with RCK/p54 are determined by different domains of AGO2, suggesting that miRNA‐mediated translational repression is unrelated to the miRISC localization at P‐body. Our continuing analysis of AGO2 deletion mutants interacting with other RISC components will have important implications for the miRNA‐mediated gene silencing.
Characterization of potential virulence factors in Rickettsia prowazekii as lysine methyltransferases
Authors: Abeykoon Amila H. , Chao Chien-Chung , Yang David C.H. , Ching Wei-Mei
Published: June 2021

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Lysine methylation of OmpB (outer membrane protein) of Rickettsia correlates with the bacterial virulence. However, no lysine methyltransferase from Rickettsia has been characterized. Bioinformatic analysis of the Rickettsia prowazekii genome revealed six potential lysine methyltransferases. Codon‐optimized cDNAs of the six potential methyltransferases were synthesized and subcloned into pET28a. The six proteins were then expressed in E. coli BL21(DE3) and purified by affinity chromatography on Ni‐NTA agarose. The methyltransferase activity was assayed by the methyl incorporation of radioactively labeled S‐adenosyl‐methionine. Two of the six putative methyltransferases showed time‐ and enzyme‐concentration‐dependent methylation of the recombinant AN and K fragments of OmpB from R. typhi. The stoichiometry of the methyl incorporation suggests methylation at multiple sites of both fragments. Western blot analysis using trimethyl‐lysine specific antibodies confirmed the trimethylation of lysines in recombinant fragments of OmpB by the methyltransferases. Enzymatic methylation of OmpB delineates the functions of these proteins, and may provide tools for the development of diagnostic reagents
The Mechanism of [PSI+] Prion Curing in Response to Mild Thermal Stress
Authors: Klaips Courtney Lee , Serio Tricia R
Published: June 2021

Prions are proteins that can adopt more than one stable conformation in vivo. Remarkably, the alternative non‐native conformers can self‐replicate their folds by forming aggregates that template the conversion of native conformers to a like state. Because these conformational changes alter the normal activity of the protein, the self‐replication process allows prions to confer new and transmissible phenotypes. In yeast, the fragmentation of prion aggregates by the molecular disaggregase Hsp104, along with its Hsp70 and Hsp40 co‐chaperones, to create new templates and to facilitate their partitioning to daughter cells is an essential step in this pathway. But the expression and activities of these and other factors mediating protein quality control are profoundly altered by external stresses. While such changes in environmental conditions have been reported to “cure” certain variants of the [PSI+]/Sup35 prion in yeast, the mechanisms by which these transitions occur are poorly understood. Here, we report that mild heat shock cures [PSI+] by dissolving existing prion aggregates. Our studies suggest that these effects are mediated not simply by the increase in chaperone expression but rather by the interplay between ordered, prion aggregates, and disordered, heat‐induced protein aggregates in the engagement of these factors. This work is supported by NIH grant F31AGO34754.
Low‐level oxidative stress increases chaperone protein activity and alters steroid receptor•hsp90 heterocomplex formation
Authors: Stone Orrin J. , Franklin Hannah R. , Murphy Patrick J. M.
Published: June 2021

The interruption and subsequent restoration of oxygenated blood flow leads to oxidative stress and localized tissue damage, known as ischemia‐reperfusion injury. Two likely intracellular components involved in protecting the cell against ischemia‐reperfusion‐related damage are the molecular chaperone proteins hsp90 and hsp70. The objective of this study was to determine the impact of oxidative stress on hsp90/hsp70 chaperone protein activity using a glucocorticoid receptor‐based in vitro reconstitution assay. We assessed the hypothesis that increasing concentrations of free radicals (0–300 μM H2O2) would stimulate hsp90/hsp70 function. We observed through western blotting and hsp90‐dependent ligand binding that the hsp90/hsp70 chaperone machinery remained catalytically active and bound to the receptor until oxidative stress levels >100 μM H2O2. Oxidative conditions exceeding 100 μM were correlated with dissociation of steroid receptor•hsp90 heterocomplexes and loss of hsp90/hsp70 functional activity. Most interestingly, there was an observed 25–40% increase in hsp90‐dependent ligand binding activity under mild oxidative stress (~1 μM H2O2). We posit a biochemical explanation for the role of hsp90 in ischemic preconditioning, where mild rounds of ischemia and reperfusion confer a protective effect within the cell against future, prolonged periods of oxidative stress.
The role of mitochondrial peptide deformylase in coordinating respiratory chain assembly
Authors: Battersby Brendan James , Richter Uwe , Lahtinen Taina , Ruotsalainen Pilvi , Myöhänen Maarit , Marttinen Paula
Published: June 2021

Translation of mitochondrially‐encoded proteins occurs on a dedicated set of mitochondrial ribosomes with mechanistic features more similar to their prokaryotic ancestors than that seen for cytoplasmic ribosomes. In the majority of eukaryotes, mitochondrial translation initiation requires a formyl‐methionine. In prokaryotes, the formal group and N‐terminal methionine are removed as part of the N‐terminal methionine excision (NME) pathway for protein stability. This pathway depends upon the initial deformylation reaction catalyzed by peptide deformylase. Mitochondria also contain this dedicated machinery for NME, and yet in vertebrates, only 1 of the 13 mitochondrial polypeptides has the formyl group and methionine removed from the mature protein. Loss of peptide deformylase activity leads to a defect in oxidative phosphorylation, yet the molecular basis is unknown. We have been investigating the function of Peptide deformylase in murine embryonic fibroblasts and discovered a novel regulatory loop for translation involving a retrograde response that accounts for the defect in oxidative phosphorylation, and ultimately affects the cellular growth rate. Details of this regulation will be presented.
In silico studies to explain the interactions to inactivate NAPDH oxidase by apocynin and its dimmer
Authors: Macías-Pérez Martha Edith , Martínez-Ramos Federico , Padilla-Martínez Itzia Irene , Correa-Basurto José , Tolentino-López Luis Esteban , Rosales-Hernández Martha Cecilia
Published: June 2021

NADPH oxidase has been considered a therapeutic target in hypertension disease due to superoxide anion (O2•‐) react with Nitric Oxide (NO) diminish its vasodilator effect. Apocynin dimer has been reported as NADPH oxidase inhibitor due to that is able to interact with the p47phox subunit and prevent the binding with the p22phox subunit, however this fact has been not reported for apocynin. For this reason a in silico study was done to know the interactions between apocynin and its dimmer with two different conformations of p47phox one in extended conformation (1NG2.pdb), and the other in close conformation (1WLP.pdb) which consist of: two SH3 domains and a polybasic region, that are important during the coupling with the p22phox subunit. The results showed that in the extend conformation apocynin is bind at the SH3B domain of p47phox meanwhile its dimmer is bind at the polybasic region maintained to p47phox in its autoinhibited form avoiding the coupling with p22phox. Groemping Y., Lapouge K., Smerdon S.J., Rittinger K. Molecular Basis of Phosphorylatión‐induced Activatión of the NADPH oxidase. Cell. 2003;343–355.
Property of the antioxidant activity using xanthine oxidase reaction
Authors: Masuoka Noriyoshi , Maeta Ayami , Kamimura Wakana , Kubo Isao
Published: June 2021

Inhibitory activity of the superoxide anion (O2−) generation catalyzed by xanthine oxidase (EC 1.1.3.22) with natural products was used to estimate the antioxidant activity. To understand properties about the activity, we examined antioxidant activity of anacardic acids, alkyl gallates and flavonoids. Xanthine oxidase consisted of several oxid‐reduction forms in the molecule. The oxidized forms catalyzed O2− generation from oxygen but the reduced forms catalyzed only hydrogen peroxide formation. We deduced that antioxidants affected three reactions, (A), (B) and (C) to indicate antioxidant activity. In reaction (A), antioxidants function inhibitors of xanthine oxidase reaction, and both uric acid formation and O2− generation were suppresses. In the reaction (B), antioxidants function as reducing reagents of xanthine oxidase, and the reduced enzyme cannot generate O2−. In the reaction(C), antioxidants function as scavengers of O2− generated by xanthine oxidase. We also indicate that effects of antioxidants on reaction (A) and (B) are stronger than those on reaction (C).
Kinetics of exoglucanase and endoglucanase produced by Aspergillus niger NRRL 567
Authors: Ahmed Sibtain , Ghori Mohammad Ishfaq , Malana Mohammad Aslam , Jamil Amer
Published: June 2021

The objective of this study was to study the kinetics of exoglucanase (EXG) and endoglucanase (EG) from Aspergillus niger NRRL 567. The optimum pH and temperature for crude EXG and EG f rom A. niger NRRL 567 was found to be 3.5 and 30 °C respectively. Zn2+, Ca2+, Mn2+, Co2enhanced EXG activity whereas Mg2+, Fe2+ and Hg2+showed various degree of inhibitory effects. Cu2+ enhanced EXG activity and inhibited EG activity. For EXG, by using the Lineweaver‐Burk plot (1/V vs 1/S), the Km value was 7.35 mg/mL and Vmax value was 1.9 μmol/mL/min. For EG, by using the Lineweaver‐Burk plot (1/V vs 1/S) the Km value was 4.75 mg/mL and Vmax value was 1.71 μmol/mL/min. The energy of activation (Ea) for EXG and EG were 21.20 kJ mol−1 and 22.52 kJ mol−1 respectively. The Q10 values obtained for the EXG and EG were 1.38 and 1.4 respectively. These enzymes had lower Km value that shows their high affinities for their respective substrates. In conclusion this study demonstrates that these enzymes are suitable for industrial use.
Determining the effect of dithiolethione compounds on the activity of human glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Chavis John Taylor , Neal Jacob , Switzer Christopher , Wink David A. , Garcin Elsa
Published: June 2021

Dithiolethione compounds have chemopreventive, cytoprotective, and antimitogenic effects. We have shown that some of these compounds interact with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). In cells, GAPDH is an abundant and multifaceted protein involved in glycolysis, cell death, DNA repair, regulation of gene expression, tRNA export, membrane fusion and transport. As a result of its plethora of roles, there is an incentive in understanding how dithiolethiones affect GAPDH activity and function. Dithiolethiones can directly or indirectly modify cysteine residues in protein targets. GAPDH contains three cysteine residues that are susceptible to thiol‐modifying agents. Therefore, we set out to examine how the dithiolethione, ADT‐OH, affects GAPDH activity.

Human GAPDH was overexpressed in bacterial cells and purified by ion‐exchange and affinity chromatography. We measured the catalytic activity of GAPDH in the absence and the presence of ADT‐OH, and in the absence of DTT. Comparison of the kinetics parameters for unmodified GAPDH and GAPDH modified with increasing concentrations of ADT‐OH reveals a concentration‐dependent decrease of GAPDH catalytic activity.

Our next step will be to determine the exact mechanism of ADT‐OH modification of GAPDH by combining site‐directed mutagenesis, mass‐spectrometry, circular dichroism, and x‐ray crystallography.

Funding

UMBC DRIF SRAIS (EG)

This research was supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program.
Structural analysis of a secretory phospholipase A2 from Clonorchis sinensis: therapeutic implications for hepatic fibrosis
Authors: Hariprasad Gururao , Srinivasan Alagiri , Kaur Punit , Kumar Manoj , Singh Tej Pal
Published: June 2021

Hepatic fibrosis is a common complication of the infection by the parasite, Clonorchis sinensis with an increased risk of cancer. A secretory phospholipase A2 enzyme (CsPLA2) from the parasite is implicated in the pathology. This is an attractive drug target necessitating its characterization. Structure modeling of the CsPLA2 was done based on the 40% homology it shares with group III bee venom PLA2. On analysis, the enzyme essentially comprises of a calcium binding loop, three helices, two sets of β‐wings and an elongated C‐terminal extension with the entire structure stabilized by four disulfide bonds. There are three significant differences between the pathogenic CsPLA2 and its house keeping human isoform (HsPLA2). They are (1) participation of His‐Asp‐Tyr in hydrogen bond formation at the active site as compared to His‐Asp, Phe arrangement in HsPLA2; (2) hydrophobic channel environment is less polar in CsPLA2 and (3) there are six cationic residues distributed across the putative membrane binding surface of CsPLA2 as compared to only three cationic residues in the latter. These features are important physiochemical parameters that not only help to understand the pathogenesis of the disease but can be exploited to design specific inhibitor molecules for a targeted therapy of liver fibrosis caused by the parasite. This research was supported by Department of Science and Technology, Govt. of India (SR/FT/18).
Development of an LC‐MS/MS based biochemical assay to evaluate Isoprenylcysteine carboxyl methyltransferase (ICMT) as a cancer target
Authors: Bingham Patrick , Maegley Karen
Published: June 2021

We developed a highly sensitive LC‐MS/MS based biochemical assay to quantitate the enzymatic activity of ICMT. The activity of endogenous ICMT was first determined in two kras dependent cancer cell lines and then following treatment with various siRNA constructs. The degree of knockdown was quantiated by comparison of siRNA treated cells with control treated cells. Enzyme activity loss correlated well with loss of messenger RNA. Time course experiments were also performed to evaluate the turnover time for endogenous ICMT in these cell lines. These data together with anchorage‐independent proliferation experiments allowed us to conclude that 80–90% loss of ICMT activity was insufficient to slow the growth of 10 different disease relevant cancer cell lines. Furthermore, ICMT knockout cell lines were engineered to evaluate whether 10–15% remaining ICMT activity was sufficient to enable cell growth. The LC‐MS/MS assay was unable to detect ICMT enzymatic activity in these cells. The lack of cell growth inhibition in these cells suggests that ICMT is not critical for kras localization and/or activity in these kras mutant cell lines. The use of LC‐MS/MS as a biochemical assay product detection method enabled us to monitor enzymatic activity to quantitate loss of protein in knockdown studies and loss of function in knockout studies. Our assay enabled the evaluation of ICMT as a Ras pathway therapeutic target.
Leishmania mexicana potential drug target
Authors: Kreiss Tamara , Patel Seema J , Goodey Nina , Siekierka John J , Sayakci Aysenur
Published: June 2021

We have been studying the Leishmania mexicana protein kinase Lmx‐MPK1. Lmx‐MPK1 is required for parasite viability in the host and represents a potential therapeutic target. We have been studying the interaction of the human p38 mitogen‐activated protein kinase (p38 MAPK) inhibitor, BIRB 796, with Lmx‐ MPK1. BIRB 796 is a unique allosteric MAPK inhibitor which binds to a highly conserved Asp‐Phe‐Gly motif within the active site of p38 MAPK. This site is conserved in other MAPKs and Lmx‐MPK1 as well. In order to assess BIRB 796 binding to LmxMPK1, we used fluorescence quenching to determine structural changes resulting from the binding event by measuring a change in tryptophan signaling at 290nm excitation and 340nm emission wavelengths. We will report on our results applying this technology to characterize kinase inhibitor binding.
Hymeglusin inhibition of bacterial hydroxymethylglutaryl‐ CoA synthase (mvaS)
Authors: Miziorko Henry M , Skaff D Andrew , Ramyar Kasra X , McWhorter William J , Geisbrecht Brian V
Published: June 2021

Hymeglusin (1233A; F244) is established as a specific beta lactone inhibitor of eukaryotic hydroxymethylglutaryl‐CoA synthase (HMGCS; Greenspan et al., PNAS (1987) 84: 7488–7492). Inhibition results from formation of a thioester adduct to the active site cysteine. Hymeglusin inhibition has primarily been studied for eukaryotic HMGCS, with little characterization of effects on prokaryotic mvaS. Hymeglusin blocks growth of Enterococcus faecalis. After inhibitor is removed from culture media, a growth curve inflection point at 3.1 hr is observed (versus 0.7 hr for uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 5‐fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme‐inhibitor thioester adduct stability and/or solvent accessibility. The E. faecalis mvaS‐hymeglusin structure (1.95 Å) reveals virtually complete occlusion of bound inhibitor in a solvent inaccessible narrow tunnel. In contrast, eukaryotic (Brassica juncea) HMGCS (Pojer et al. (2006) PNAS 103:11491–11496) binds hymeglusin in a more solvent exposed cavity. NIH AI071028, AI090149; Marion‐Merrell‐Dow Fdn.
Mutational activation of PI3′‐kinase‐α dramatically accelerates BRAFV600E or KRASG12D‐induced lung tumorigenesis
Authors: Trejo Christy Lee , Greenberg Shon , Marsh Vicky , Phillips Wayne , McMahon Martin
Published: June 2021

Lung cancer is responsible for ~160,000 deaths/year in the USA. Activation of signaling pathways downstream of receptor tyrosine kinase is common in non‐small cell lung cancer (NSCLC). Lung epithelium specific expression of mutationally activated KRASG12D or BRAFV600E leads to tumorigenesis. Unlike KRASG12D, BRAFV600E expression leads to benign lung adenomas that do not progress. One explanation might be the lack of PI3′K signaling in BRAFV600E–driven tumors. We generated mice carrying Cre‐activated alleles of BRaf, Pik3ca, or both. Intranasal instillation of Ad‐Cre to initiate PIK3CAH1047R expression failed to elicit lung tumors while expression of BRAFV600E resulted in abundant benign lung tumors. However, Ad‐Cre mediated expression of both BRAFV600E and PIK3CAH1047R resulted in increased number and size of lung tumors compared to control BRafCA littermates. Pharmacological inhibition of AKT prevented the increased tumor burden observed in BRafCA; Pik3caHR mice, indicating that cooperation is AKT dependent. We treated compound KRasLSL; Pik3caHR mice with Ad‐Cre, and these mice succumbed to lung tumorigenesis significantly more rapidly than control KRasLSL littermates. These data suggest that expression of PIK3CAH1047R alone is insufficient to promote lung tumorigenesis, but cooperates in dramatic fashion with either mutationally activated BRAFV600E or KRASG12D.