bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapidly Evolving MicroRNAs Retain Their Targets by a Co‐Evolution Mechanism
Authors: Ramaiah Madhuvanthi , Shum Eleen Y. , Wilkinson Miles F. 
Published: June 2021

MicroRNAs (miRNAs) are small regulatory molecules with diverse roles in many biological systems. Most studies to date have focused on the function of conserved miRNAs. In contrast, little is known about rapidly evolving miRNAs despite their preponderance in some biological systems, including the reproductive tract. Here, we asked whether rapidly evolving miRNAs have changed their target repertoire in different species or whether they have co‐evolved with their targets to maintain regulation. As a model system to address this question, we used a set of rapidly evolving X‐linked miRNA genes selectively expressed in the testis: the X‐mir1 gene cluster. Using reporter analysis, we identified several targets regulated by both human and mouse X‐miR1 family members despite their having little or no sequences in common. Intriguingly, 3 of the 5 targets we identified with this characteristic encode translation regulatory factors—FMRP, eIF4e and CYFIP1—suggesting that the X‐mir1 cluster has a role in translational regulation in male germ cells. We found that multiple members of the X‐miR1 cluster regulated these targets, suggesting that this miRNA cluster acts as a functional unit. We propose that rapidly evolving miRNAs create a flexible regulatory environment, but at the same time, they retain crucial targets through co‐evolutionary mechanisms to maintain their physiological roles.Study supported by NIH.
Specific microRNA (miRNA) species regulate the expression of the beta‐site APP‐cleaving enzyme (BACE1) in relevant human cell types with implications for Alzheimer disease
Authors: Lahiri Debomoy K , Long Justin M 
Published: June 2021

MiRNAs are small RNAs that mediate potent inhibitory effects on gene expression and which recent studies implicate in the regulationof gene products involved in Alzheimer's disease (AD) (Long & Lahiri, Exp. Neurol., 2011). Aberrations in AD may result from the over‐production of amyloid‐β peptide (Aβ), a product of Aβ precursor protein (APP). Dysregulation of proteins involved in Aβ production, such as APP and BACE1, may contribute to excess Aβ deposition. Using multiple bioinformatic tools and functional studies, we reported a specific microRNA (miR‐101) that regulates APP levels (Long & Lahiri, BBRC, 2011). Here we report the discovery of a novel BACE1‐specific miRNA regulatory interaction. First, we prepared a BACE1 3′‐UTR reporter construct by inserting the 3.9 kb BACE1 3′‐UTR downstream of a Renilla reporter luciferase gene. The reporter construct was delivered along with several miRNAs predicted to target the BACE1 3′‐UTR into human cultures. Several “hits” (e.g. miR‐339‐5p) resulted in reduced reporter expression. We validated the reporter expression for miR‐339‐5p by Western analysis of native BACE1 levels, which were significantly reduced following miR‐339‐5p delivery (with anticipated reductions in toxic Aβ levels). Our results reveal a novel regulatory interaction between BACE1 and specific endogenously expressed miRNA species that may serve as putative therapeutic targets for AD.This work is supported by grants from Alzheimer's Association and NIH to Dr. D.K. Lahiri.
Deletion of the Cathepsin B gene improves memory deficits in a Alzheimer's disease mouse model expressing APP containing the wild‐type β‐secretase site sequence
Authors: Hook Vivian , Yu Jin , Zhu Hong , El-Amour Salim , Hook Gregory , Kindy Mark 
Published: June 2021

Severe memory loss is detrimental in Alzheimer's disease (AD). We hypothesize that cathepsin B (CatB) participates in memory deficits of AD. Therefore, goal of this study was to assess the effects of cathepsin B gene knockout (CatB) on the memory deficits of a transgenic AD mouse model expressing human APP containing the wild‐type (WT) β‐secretase site and the London mutant γ‐secretase site sequences (AβPPWT/Lon). Results show that CatB gene knockout in AβPPWT/Lon mice results in significant improvement in memory, assessed by the Morris water maze test. Moreover, the CatB gene knockout reduces brain leevels of amyloid‐β peptides (Aβ) and carboxyl‐terminal β‐fragment (CTFβ); these data are consistent with CatB possibly having a role as β‐secretase for the WT β‐site of AβPP expressed in the majority of AD patients. In contrast, deleting the BACE1 gene in the AβPPWT/Lon mice had no effect on memory deficits or on the biomarkers. In conclusion, these data validate CatB as a drug target for improving memory deficits in AβPPWT/Lon mice and suggest that CatB inhibitors may be effective for improving memory in the majority of AD patients.This work was supported by NIH grants R21AG0311963, R44AG030865, and R44AG032784 to GH, and a grant from the Alzheimer's Association to VH.
A Comprehensive Analysis of Lysine Acetylation in Ribosomal Proteins
Authors: Koc Hasan , Cimen Huseyin , Stallard Adam , Koc Zeynep C , Koc Emine C 
Published: June 2021

Protein synthesis is one of the most conserved processes in living organisms; specifically, the conservation between mammalian mitochondrial and bacterial translation machineries/ribosomes has been the most surprising one of all. Our comprehensive studies of post‐translational modifications (PTMs) to ribosomal proteins and large‐scale mass spectrometry (MS)‐based PTM mapping studies from several other laboratories suggest that the mitochondrial and bacterial ribosomes are highly modified by acetylation at their ε‐amino groups of Lys residues. The majority of the modified proteins were found to be located in the essential regions of ribosome function including, mRNA‐ and tRNA‐binding path, peptide exit tunnel, and the L1 and L7/L12 stalks. Interestingly, not only the proteins with conserved homologous functions but also the conserved residues are post‐translationally modified in both systems. The nature of this modification in both mitochondrial and bacterial ribosomes indicates that it is an essential and evolutionarily conserved mechanism for the regulation of protein synthesis.Supported by NIH grant R01GM071034 (E.C.K.).
Kinetic analysis of β‐Galactosidase activity from Thermococcus kodakarensis KOD1, an extreme thermophile
Authors: Hwa KuoYuan , Subramani Boopathi , Shen Santai , Lee Yu-May 
Published: June 2021

The enzyme β‐galactosidase catalyzes the hydrolysis of lactose into glucose and galactose. An extracellular β‐galactosidase of 489‐amino acid length and 57‐kDa molecular weight monomer was isolated, expressed and purified from an extreme thermophile, Thermococcus kodakarensis KOD1. Here, the enzyme kinetic parameters of the β‐Galactosidase were analyzed through the hydrolysis of the yellow chromogenic substrate, chlorophenol red β‐D‐galactopyranoside (CPRG). Ad, the red colored chlorophenol red product was measured. The optimum pH and optimum temperature for the enzyme activity were 6.5 and 100°C. The maximum enzyme activity was retained even at 110°C, the highest temperature used in the study. The kinetic parameters Km and Vmax of the purified enzyme were 1.4mM and 2.5 U/mg for CPRG at the optimum pH and temperature conditions. The enzyme could not be inhibited with glucosidase inhibitors.The research was supported by the National Taipei University of Technology to KYH (NTUT 100‐140‐01, NTUT 99‐140‐04) and by Academia Sinica to YML.
ATP alters IKK‐2 inhibitor affinity for IKK2 enzyme and reveals non‐equivalent inhibitor binding sites
Authors: Liu Jia Yeu , Stevens Kirk , Mourey Robert 
Published: June 2021

MethodInhibitor off‐rate was measured by two methods: (1) pre‐binding the enzyme with inhibitor followed by measuring the recovery of enzyme activity upon a 200‐fold dilution of the enzyme‐inhibitor complex in the presence of 1mM ATP and (2) measuring the shift in the compound potency (IC50) curves obtained by prebinding the enzyme and inhibitor in the absence of ATP followed by addition of either 1μM or 1mM ATP.ResultsThree literature inhibitors with potency (IC50) ranging from <1 to 35 nM were used for this study: PHA‐408 (Pfizer), GSK‐082 (GSK), and AVE‐002 (Aventis). The off‐rates (T1/2) measured by the dilution method are 124 and <15 min for PHA‐408 and GSK‐082, respectively. The off‐rate for AVE‐002 was biphasic with half of the enzyme‐inhibitor complex dissociating immediately (T1/2 < 2 min) and the other half bound tightly (T1/2 54 min). The IC50 curves at 1μM and 1mM ATP for PHA‐408 showed no shift using a short 2 min assay and is consistent with the slow off‐rate. GSK‐082 showed a > 10 fold loss of potency with 1mM ATP, as expected for a fast off‐rate inhibitor. AVE‐002 IC50 curve with 1μM ATP was monophasic but displayed a biphasic response with 1mM ATP.ConclusionATP can change the off‐rate of IKK‐2 inhibitor bound to the enzyme. Additionally, high ATP revealed that AVE‐002 bound in the two inhibitor binding sites of the IKK‐2 homodimer non‐equivalently.
Identifying new drug targets against African Trypanosomiasis
Authors: Kalidas Savitha , Phillips Margaret 
Published: June 2021

Identifying new drug targets against African Trypanosomiasis Human African Trypanosomiasis (HAT), one of the common tropical neglected diseases (NTDs), is responsible for extensive suffering, death and economic burden in developing countries. NTDs represent the sixth greatest global health burden in terms of the disability adjusted life years. Research and development yielding safe and effective drugs for their treatment and prevention has been recognized as a global health priority. Current drug therapies for HAT are inadequate, hard to administer and have increasing drug resistance problems. To identify and validate new targets for drug discovery we, in a coordinated effort from several groups, are systematically analyzing approximately 200 genes coding for enzymes involved in different biochemical and signaling pathways by RNAi knockdown or gene knockout. We have recently reported the development of a new vector (pTrypRNAiGate) derived from pLEW100 that utilizes the Gateway® recombination system to facilitate easy production of hairpin RNA constructs which facilitates high‐throughput applications for gene silencing and provides a tool for functional genomics in T. brucei. Using this vector we have knocked down 7 aminoacyl tRNA synthetases (AARS), which have an important role in protein translation. RNAi against 5 AARS resulted in growth inhibition of T.brucei bloodstream form in vitro. Two of them, Prolyl tRNA synthetase and Asparagyl tRNA synthetase show morphology changes upon RNAi induction suggesting cell cycle defects. Further validation of AARS, recombinant protein production and assay development is in progress. This work contributes toward a systemic analysis of T. brucei enzymes to identify and validate new drug targets against the infectious disease, African Trypanosomiasis.NIH Grant: AI078962
Arachidonic acid inhibits retinoic acid‐induced neuritogenesis of human neuroblastoma SH‐SY5Y cells
Authors: AMISSAH FELIX , AYUK-TAKEM LAMBERT T. , LAMANGO NAZARIUS S. 
Published: June 2021

Polyisoprenylated proteins regulate intracellular processes such as cell growth, differentiation, oncogenic transformation and actin cytoskeletal organization. Recent discoveries show that polyunsaturated fatty acids (PUFAs) may be involved in neurodegenerative diseases. PUFAs such as arachidonic (AA), and eicosapentaenoic acids (EPA) induce apoptosis in cultured cells. This effect has recently been shown to correlate with the inhibition of polyisoprenylated methylated protein methyl esterase (PMPMEase). We propose that abnormally high levels of PUFAs may be deleterious to neuronal cells. Enzyme kinetics analysis with PMPMEase revealed a 2‐fold decrease in KM from 16.2 ± 1.34 to 8.60 ± 1.20 and 7.84 ± 1.02 μM in the presence of 10 μM AA or EPA, respectively. A relatively severe degenerative effect of AA on SH‐SY5Y cells was observed in the absence of 10% FBS (EC50 of 6.2 μM). AA (2 – 10 μM) caused concentration and time‐dependent inhibition of neuritogenesis in retinoic acid (RA)‐differentiated SH‐SY5Y cells. Significant decreases in the percentage of neurite‐bearing cells and neurite lengths were observed after 24 h with 10 μM AA. These results suggest that excessive PMPMEase inhibition may extend beyond controlling cell viability to the degeneration of vulnerable cells such as neurons.Supported by NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020).
AN INTEGRATED EXPERIMENTAL WORKFLOW TO INCREASE THROUGHPUT AND DATA ROBUSTNESS FOR ANALYSIS OF MAMMALIAN PROTEIN INTERACTION NETWORKS
Authors: Auerbach Daniel , Bruderer Roland , Uhlmann Thomas , Rezwan Mandana 
Published: June 2021

To understand the organization of protein complexes and how biological information is propagated via protein‐protein interaction is becoming increasingly important to get new functional insights on cellular processes. Affinity purification coupled to mass spectrometry (AP‐MS) has been successfully used in the past to characterize protein complexes. However, mainly due to significant experimental limitations the success and power of the AP‐MS strategy has been underrated to date. Here, we describe an integrated workflow for characterization of mammalian protein complexes. The experimental pipeline comprises rapid generation of isogenic mammalian cell lines, protein complex analysis using an efficient double affinity purification strategy followed by state‐of‐the‐art nano‐LC‐Orbitrap‐MS analysis.We use this pipeline to study interactions associated with BMP signaling systems. In particular we focus on ACTR‐1/Alk2 and FKBP12; two proteins intimately linked to Fibrodysplasia Ossificans Progressiva (FOP) a catastrophic human disorder leading to heterotopic ossification throughout the body.The high performance of our strategy is well suited to identifying protein interactions partners that can subsequently be used as (1) entry points for focused research projects and (2) to more globally map out whole signaling systems linked to important biological processes.
Characterization of the Effects of Lipid‐binding on the Chaperone Activities of 70‐kDa Heat‐Shock Proteins
Authors: McCallister Chelsea Marie , Nikolaidis Nikolas 
Published: June 2021

Hsp70s are a group of molecular chaperones essential for cellular homeostasis and survival. Apart from their indispensable intracellular functions, Hsp70s are also found in close proximity to biological membranes and the extracellular environment, where they activate the immune system and function in cell signaling. It is also known that Hsp70s interact and bind to the primary constituent of membranes, the lipids. However, it is currently unknown whether lipid‐binding affects their chaperone functions. To determine the mechanistic role that lipid‐binding plays on the function of Hsp70s as molecular chaperones we tested the effects of lipid‐binding on the interaction of Hsp70s with nucleotides and protein substrates, as well as whether lipid‐binding affects their refolding abilities. Our results show that lipid‐binding is altered when HSPA1A binds to nucleotides, while it remains unaffected when the protein binds to protein substrates. Furthermore, we show that lipid‐binding significantly reduces the refolding activity of HSPA1A. Taking into account the fact that lipid‐binding is mediated by the nucleotide‐binding domain of the protein, we speculate that a specific conformational change caused by the lipid‐binding reduces the ATPase activity and subsequently the refolding activity of the chaperone. These data strongly suggest that lipid‐binding regulates the chaperone activities of Hsp70s.This project was supported by funds from CSUPERB and CSUF to NN.
Investigations of the Folding Dynamics of the RNA Pseudoknot Structural Motif via Massively Parallel Molecular Dynamics
Authors: Pham Benjamin D. , Kusaba Carolyn C. , Bakhom Mona , Wang Richard , Sorin Eric J. 
Published: June 2021

RNA pseudoknots compose a three‐dimensional structural motif that is present in the catalytic cores of some ribozymes, and are also capable of stimulating ribosomal frameshifts. Furthermore, their complex topology and non‐canonical hairpin‐loop composition make pseudoknots an essential structural motif with which to study the RNA folding process. Here we report our analysis of nearly 20,000 independent all‐atom molecular dynamics simulations of the ribosomal frame‐shifting pseudoknot of Luteovirus and the tmRNA pseudoknot from Aquifex aeolicus, which share global topology but have only ~50% sequence similarity. Using the Folding@Home distributed computing network and our new Pathway Enumeration sampling method, a cumulative sampling time of over 100 μs was achieved for each of these pseudoknots. K‐means clustering was used to identify 27 conformational microstates for each pseudoknot and similar folding pathways were identified for these two sequences. In agreement with our earlier work, this study suggests that native state topology is a predominate factor in the RNA folding mechanism.This work was made possible by the worldwide Folding@Home volunteers who contributed invaluable processor time, Women & Philanthropy, and a Research Corporation Cottrell College Science Award.
J‐protein requirements of multiple [PSI+] prion variants in two distinct yeast strains
Authors: Hines Justin Keith , Patel Milan J 
Published: June 2021

The objective of this study was to determine the importance of prion variant and yeast strain variation on prion chaperone requirements. The J‐protein Sis1 and the prion [PSI+] were used as a model system to explore the impact of amyloid conformation (prion variants) and yeast strain polymorphisms (yeast genetic background) on chaperone requirements. The primary method used in this study was complementation by plasmid‐shuffling in the budding yeast Saccharomyces cerevisiae.Summary of ResultsYeast strain background had a small but noticeable effect on chaperone requirements; this effect is likely due primarily to altered chaperone protein levels between strains. While many prion variants shared similar chaperone requirements, some significant differences were found.ConclusionsJ‐protein chaperone requirements are significantly determined by the specific amyloid conformation of a yeast prion but relatively unaffected by yeast genetic background. Funding for this research was provided by Lafayette College in the form of the EXCEL Scholars program, Department of Chemistry research funds, and start‐up funds provided for JKH.
Reprogramming of Adult Skin Cells to Pluripotency in Human and Dolphin
Authors: Federico Tessya , Federico Niksha , Sternik Gabriel 
Published: June 2021

Bottlenose dolphins (Tursiops sp.) typically survive deep soft tissue injuries from shark bites via an impressive, less understood, wound healing process which in comparison to humans is rapid (days vs. weeks), efficient (no need for external intervention), and has a much higher success rate (little if any scarring while completely regenerates damaged tissues).In this study, directed reprogramming of fibroblasts into pluripotent stem cells (iPSC) in humans and dolphins was compared. Molecular and Cellular analyses were designed to determine whether mammalian stem cell pluripotency‐inducing factors are also involved in inducing pluripotency in skin fibroblasts obtained from bottlenose dolphins possibly contributing to the observed efficient and rapid wound healing and tissue regeneration in these animals.Our results indicate that dolphin skin fibroblasts are much more efficiently reprogrammed to induced pluripotency in‐vitro. The presence of an inherent in‐vivo reprogramming process unique to dolphin fibroblasts (absent in human fibroblasts) that may be involved in the observed efficient wound healing process in dolphins was also detected. Further understanding of Pluripotency inducing intracellular pathways and their triggering mechanisms should enable better understating of the similarities and differences between stem cell mediated wound healing in dolphins and human.
Using protein modification‐based networks to explore cellular signaling and biological function
Authors: Kornhauser Jon M , Tkachev Sasha , Zhang Bin , Skrzypek Elzbieta , Murrray Beth , Latham Vaughan , Hornbeck Peter V 
Published: June 2021

PhosphoSitePlus™ (PSP) is an open proteomics resource that provides manually‐curated information about published sites of protein post‐translational modification (PTMs). Over 100,000 published PTM sites are currently represented in PSP. Several different classes of experimental information about each PTM site are aggregated in PSP, including enzymes (e.g., protein kinases) that are reported to catalyze the modification; ligands and pharmacological agents that modulate the modification; and consequences of a modification, including effects on protein‐protein interactions; effects on the function of the modified protein; and consequences for biological processes, including relevance of the modification to normal and disease states.The rich and extensive data in PSP can be exploited to assemble signaling networks to explore cellular pathways that regulate biological processes including disease. We have used the Cytoscape platform to construct networks representing the data in PSP. Because PSP is unique among proteomic resources in that it contains data that is PTM site‐centric, rather than protein‐centric, analysis of these networks may provide novel insights into cellular signaling.We are employing graphical and statistical approaches, including correlation and cluster analyses, to explore these networks. Our expectation is that the increased resolution of our site‐specific networks, compared to more typical analysis using protein‐centric networks, may allow us to detect novel signaling nodes that are important for specific biological outcomes.www.phosphosite.org
Oral administration of transgenic Dp2 peptide milk induces tolerance from allergic asthma
Authors: Chen Chuan-Mu , Pai Shun-Yuan , Chen Hsiao-Ling 
Published: June 2021

ObjectiveWe aimed to study whether oral administration with transgenic milk expressing a dust mite allergen can down‐regulate the allergen‐specific airway inflammation.MethodsThe transgenic CD‐1 mice expressed recombinant group 2 allergen of Dermatophagoides pteronyssinus (Dp2) in their milk were generated through recombinant genetic technique. In animal model, the pups were fed with transgenic Dp2‐containing milk or wild‐type (WT) milk, respectively.ResultsUpon sensitization and challenge, mice fed with transgenic milk had decreased T‐helper 2 (Th2) response and increased T‐helper 1 (Th1) response in the airway when comparing with mice fed with WT milk. Moreover, pre‐treatment with transgenic Dp2‐milk make attenuated airway inflammation and decreased airway hyper‐responsiveness.ConclusionIn animal model, oral administration of transgenic milk expressing Dp2 allergen was demonstrated to have tolerance induction and protection from allergic airway inflammation.