bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining the role of SREBPs in Viral Infection
Authors: York Autumn Gabrielle , Bensinger Steven J
Published: June 2021

Over 180 million people are infected with Hepatitis C Virus (HCV). Chronic HCV infection can cause liver cirrhosis and cancer, and is the primary indication for liver transplant. To date, there is no vaccine and only limited treatment options for HCV. Several studies have noted alterations in host lipid metabolism in response to HCV infection. The Sterol Regulatory Element Binding Proteins (SREBPs) are the master transcriptional regulators of de novo lipid biosynthesis. SREBPs regulate upwards of 40 genes involved in lipid biosynthesis, lipoprotein import and intracellular transport, including the rate limiting enzymes in both cholesterol and fatty acid synthesis (HMG‐CoA Reductase and Acetyl‐CoA Carboxylase, respectively). Interestingly, many SREBP target genes are known to be upregulated in response to HCV infection. To better understand the importance of SREBP on HCV biology, we stably modified Huh 7.5.1 liver cells with shRNA against the 2 major isoforms of SREBPs (SREBP1 and 2) and SCAP, a chaperone protein required for SREBP activation (Loss‐of‐function SCAP acts as a double knockdown of SREBP1 and 2). Preliminary studies using immunofluorescence imaging and qPCR indicate significant attenuation of HCV lifecycle in response to loss‐of‐function SREBP1 and SREBP2. Markedly, loss‐of‐function SCAP attenuates HCV lifecycle to an even greater extent, suggesting that both SREBP1 and SREBP2 target genes are required for efficient HCV lifecycle. Conversely, gain‐of‐function of either SREBP1 or SREBP2 results in 20 to 30‐fold increase in viral replication, respectively. Our data provide strong evidence that SREBPs play a critical role in supporting HCV infection. In the future, we aim to define the mechanism by which SREBP and HCV interact on both a cellular and molecular level. Furthermore, our work will evaluate SREBP activation as a potential novel pharmacologic target for chronic HCV infection.
Fatty acids and their thioester derivatives as ligands for human PPARα – A comparison to murine PPARα
Authors: Oswal Dhawal Pravin , Balanarasimha Madhumitha , Kaliappan Alagammai , Rider S. Dean , Hostetler Heather
Published: June 2021

Peroxisome proliferator activated receptor alpha (PPARα) belongs to the family of the ligand dependant nuclear transcription factors that play key roles in regulating lipid homeostasis. Previous studies have suggested binding of mouse and xenopus recombinant PPARα to fatty acids (FA) and their thioester derivatives (fatty acyl coenzyme A; FA‐CoA) however, no such studies have been done with full length human PPARα (hPPARα). Owing to differences in the physiological effects of PPARα activation across species (i.e. peroxisome proliferation seen in rodents but not in humans), it is possible that binding affinities and specificities for such ligands may differ. Dysregulation of FA and FA‐CoA may alter the transcriptional activity of PPARα and may contribute significantly to the pathogenesis of the metabolic syndrome. In the current study, we test the binding of various FA and FA‐CoA to hPPARα. Data from both spectrofluorometry and circular dichroism spectroscopy suggest binding of both saturated and unsaturated FA and FA‐CoA to the hPPARα at physiologically relevant concentrations found inside the cell. This is in contrast to murine PPARα which bound to only unsaturated FA but not saturated ones. Our data helps determine various endogenous ligands for hPPARα which will further help delineate the role of PPARα as a nutrient sensor in regulating energy homeostasis. This work is supported by USPHS NIH grant DK77573.
Phosphorylation of Prdx6 mediated by MAPkinase induces conformational change with concomitant increase in its phospholipase A2 activity
Authors: Rahaman Hamidur , Zhou Suiping , Dodia Chandra , Shuvaeva Tea , Feinstein Sheldon I. , Fisher Aron B
Published: June 2021

Peroxiredoxin 6 (Prdx6), a unique non‐seleno mammalian peroxidase, is a bifunctional protein with GSH peroxidase and phospholipase A2 (PLA2) activities. We have shown that phosphorylation of Prdx6 by mitogen activated protein kinase at Thr177 increases its PLA2 activity and broadens its pH‐activity spectrum (Biochem. J. 419:669, 2009). We now have investigated the structural changes in Prdx6 protein after phosphorylation and evaluated the kinetics of its interaction with unilamellar liposomes (DPPC/egg PC/cholesterol/PG; 50:25:15:10, mol/mol) using fluorescence and circular dichroism (CD) assays. Phosphorylation of Prdx6 (pPrdx6) showed a decrease in negative far‐UV CD, increase in ANS binding and no rigid tertiary structure; compatible with a change in conformation to that of a molten globule. By isothermal titration calorimetry (ITC), pPrdx6 at pH 7.4 bound to unilamellar liposomes with much higher affinity in comparison to native Prdx6; binding of pPrdx6 to liposomes was exothermic (ΔH = −31.49 ± 0.22 kcal mol−1). Correlating our conformation studies with the published crystal structure of oxidized Prdx6 (Nat. Struct. Biol. 5:400, 1998) suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Since binding of the enzyme to the phospholipid substrate interface is a requirement for PLA2 activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhanced enzymatic activity at pH 7.4
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.
Characterization of the lipid‐binding sites of HSPA1A, a member of the 70‐kDa heat shock protein family
Authors: Donnelly Gabrielle , McCallister Chelsea , Le Dat , Nikolaidis Nikolas
Published: June 2021

Hsp70s are a family of molecular chaperones whose functions in protein folding, synthesis, and transport are essential in maintaining cell homeostasis and survival. Although members of this family are localized in specific compartments within the cell, Hsp70s have also been found at the plasma membrane and in the extracellular milieu where they function in cell signaling and membrane stability. Recruitment, translocation, anchorage, and secretion of an Hsp70 are dependent on the chaperone's ability to bind to specific lipids at the target membrane, but the precise mechanisms of these interactions are unknown because the location of Hsp70‐lipid binding has yet to be characterized. Previous experiments with HSPA1A determined that the nucleotide‐binding domain (NBD) mediates most of the lipid‐binding. Through sequence alignments and protein modeling, we identified regions that have properties characteristic of known lipid‐binding domains. Two double mutations that neutralize positively‐charged amino acids on the NBD reduced binding to several lipids. Further experiments are being performed to identify additional residues that are responsible for binding all lipids. These results provide the basis towards elucidating the mechanism by which Hsp70s interact with membranes and regulate cell survival under stress and physiological conditions. This project was supported by funds from CSUPERB and CSUF to NN.
Assessing Aggregation and Membrane Binding of LLO Domains
Authors: Huang Qiongying , Wang Yan , Roberts Mary
Published: June 2021

Listeriolysin O (LLO), as one of the major virulence factors of intracellular pathogen Listera monocytogenes, assists in the escape of the bacterium from the phagosome by specifically forming oligomeric transmembrane β‐barrels on the target membrane. Its unique acidic pH optimum lessens the toxicity towards the host cell. Cholesterol is claimed to be the membrane receptor of LLO with the C‐terminal domain 4 (D4) involved in the binding and recognition. In contrast, domains 1 to 3 (D1‐3) are critical for monomer aggregation on the membrane. To better investigate these two distinct interactions with the membrane, we have separately expressed domains D4 and D1‐3 and compared their behavior to full‐length LLO using a variety of biophysical approaches (TEM, FCS, NMR). Full‐length LLO is lytic only towards cholesterol‐containing membranes and at acidic pH. D4 binds to membranes regardless of cholesterol or pH, presumably due to the hydrophobicity of its tip (ECTGLAWEWWR). However, this domain can be stabilized in a well‐folded form in DPC and SDS micelles; these have sufficient resolution in 15N‐1H HSQC experiments for some structural work. D1‐3 is nonlytic and soluble, but it aggregates significantly with the aggregation enhanced at acidic pH. These results are used to propose a modified model for the action of LLO on target membranes. These studies were supported by the NIH (GM60418).
Thermodynamic Characterization of a Riboflavin‐Dendrimer Platform for Targeted Drug Delivery Applications
Authors: Witte Amanda B. , Timmer Christine M. , Choi Seok Ki , Orr Brad , Holl Mark Banaszak , Baker James , Sinniah Kumar
Published: June 2021

We describe a biophysical characterization of generation 5 poly(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer targeting platform. Two series of dendrimers conjugates were designed, each presenting the riboflavin ligand attached at a different site (N‐3 at isoalloxazine and N‐10 at D‐ribose) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) studies were performed to investigate the binding activity for riboflavin binding protein (RfBP) in a cell‐free solution. The ITC study shows that the dendrimer conjugates have KD values of ≥465 nM on a riboflavin basis, an affinity ~ 93 fold lower than that of free riboflavin. Of the two conjugate series, the riboflavin linked at the N‐3 position showed greater affinity towards RfBP. The binding affinity was also found to decrease with increasing valency of RF. Our studies demonstrate the need to understand and better design an optimized geometry for RF‐dendrimer conjugates.
Synergistic anti‐tumor effect by a combination treatment with the dietary flavonoid luteolin and the chemotherapy drugs Tasigna or Adrucil in human pancreatic cancer cells
Authors: Aksamitiene Edita , Achanta Sirisha , Kiyatkin Anatoly , Hoek Jan B
Published: June 2021

Effects of luteolin (LU) alone or in combination with chemotherapy drugs Tasigna (nilotinib) or Adrucil (5‐fluorouracil) were explored in Bcl‐2(+) (PL5, MiaPaCa‐2) and Bcl‐2(−) (Panc1, Capan‐2) pancreatic cancer cells. Parameters of cell growth, viability, responses to growth factors and resistance to anti‐cancer agents were deteremined. Growth inhibition dose‐response curves following 72 h exposure to LU, Tasigna, Adrucil or DMSO control in 1% FBS‐containing medium were generated by fluorometric AlamarBlue assay. LU downregulated basal levels of c‐Src, FAK, STAT3 and Akt activities, reduced Bcl‐2, cyclin D1 and vimentin expression, activated Rb protein and promoted caspase‐3 and PARP cleavage in a dose‐dependent manner as shown by immunoblotting analysis. Restoration of Akt phosphorylation by PP2A inhibition did not rescue cells from LU‐induced apoptosis, which occurred in a c‐Myc‐dependent manner and was further augmented by MAPK inhibition in Bcl‐2(−) cells. By contrast, c‐Myc inhibiton increased LU‐induced apoptotic signals in Bcl‐2(+) cells. The ability of LU to synergize with drugs to accelerate PARP cleavage was cell type‐dependent and more pronounced in IGF‐1‐treated cells, suggesting that EGF engaged compensatory survival mechanism(s) mediated by ERK1/2. LU may yield additional benefit in combined chemotherapy applications in preclinical in vivo studies of pancreatic cancer.
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.
Molecular Recognition of Metal Ions by Bacterial Ribonuclease P
Authors: Liu Xin , Fierke Carol Ann
Published: June 2021

Ribonuclease P (RNase P) catalyzes the 5′ end maturation of precursor‐tRNAs (pre‐tRNAs). Bacterial RNase P is composed of one catalytic RNA subunit and one protein subunit important for molecular recognition. Like many other large ribozymes, divalent metal ions both stabilize the folded structure and enhance the catalytic activity of RNase P. Identification of the RNA moieties involved in metal‐binding and discerning the function of these interactions is an important outstanding question in RNA catalysis field. The recently solved crystal structure of bacterial RNase P [1] proposed that a catalytic metal coordinates the carbonyl oxygen of the bulged uracil in the conserved helix P4. To examine this proposal, we substituted the bulged uridine in the P4 helix of circularly permuted Bacillus subtilis RNase P with 4‐thio‐uridine which substitutes a sulfur atom for the carbonyl oxygen at position 4 on the base. This substitution decreases the single‐turnover cleavage rate by 30‐fold at pH 5.5 but has little effect on the K1/2 for activation by Mg(II) in the presence of Ca(II). Furthermore, the activity of the 4‐thio‐U mutant is not rescued by the addition of manganese ions. These data argue against the formation of an inner sphere contact between the carbonyl oxygen in the bulged uridine of P4 and a catalytic metal ion in RNase P. (This work is supported by funding from NIH (GM 55387)).
Utilizing Two‐Photon Imaging and Tracking Algorithm to Study TGF‐beta1‐Regulated SH3GLB2 Protein Assembly
Authors: Sie Yong-Da , Chen Shean-Jen , Chang Nan-Shan
Published: June 2021

Human Spas‐1 ortholog SH3GLB2 (or Endophilin B2) is frequently overexpressed in metastatic prostate cancer cells. SH3GLB2 protein has a dimerization BAR domain. Here, we determined whether TGF‐β1 induces self polymerization of SH3GLB2 in cells. COS‐7 fibroblasts were stably transfected with a green fluorescent protein (GFP)‐tagged SH3GLB2 expression construct, followed by analyzing the kinetics of protein assembly using our homemade Femtosecond Laser Scanning system, which allows determination of two‐photon excitation images of GFP with spacial and temporal resolutions during time‐lapse analysis. We combined the optical flow and sum of absolute difference (SAD) methods for determining the motion and the effective velocity of the protein cluster formation. TGF‐β1 was shown to induce SH3GLB2 to undergo self‐assembly into a crescent shape, followed by cluster formation. ATPγS, an inhibitor of ATP at 20 μM, decreased the effect of the TGF‐β1 on SH3GLB2 cluster formation. The observation indicates that energy is needed for SH3GLB2 self assembly. (Supported by National Science Council to SJC and NSC, and National Health Research Institute, Taiwan and Department of Defense, USA to NSC)
Restoring Epilepsy‐Associated Mutant GABAA Receptor Proteostasis
Authors: Di Xiaojing , Mu Tingwei
Published: June 2021

GABAA (γ‐aminobutyric acid type A) receptors are major inhibitory neurotransmitter ligand‐gated ion channels in the mammalian central nervous system. Recently, several mutations in the GABAA receptors were identified to compromise protein folding, subjecting channel proteins to endoplasmic reticulum (ER)‐associated degradation, leading to epilepsy due to loss of channel function. One prominent example is the A322D mutation in the alpha1 subunit of GABAA receptor, resulting in juvenile myoclonic epilepsy. We employed a mass‐spectrometry based proteomics approach to quantify the proteome difference between wild type and alpha1(A322D)beta2gamma2 GABAA receptors. Our preliminary result showed that critical ER chaperones, namely BiP and calnexin, interact differently with alpha1 subunit of GABAA receptors. Overexpression of BiP and calnexin increased the expression level of mutant GABAA receptors due to enhanced ER folding capacity. We propose that restoring GABAA receptor proteostasis may provide a novel therapeutic strategy for the amelioration of epilepsy and perhaps other misfolding‐associated channelopathies.
Lectin–Like Ubiquitin Ligases Degrade Alpha‐1 Antitrypsin‐Z
Authors: Glenn Kevin A , Wen Hsiang , Dankle Geoff
Published: June 2021

Alpha‐1 antitrypsin deficiency is the leading cause of childhood liver failure, is responsible for two percent of all cases of emphysema in adults, and is one of the three most common lethal genetic diseases in Caucasians. The term A1AT‐Z deficiency is a misleading term; mutant A1AT‐Z fails to fold correctly and accumulates in the endoplasmic reticulum of the liver resulting in hepatic scarring, fibrosis, and carcinoma. The failure to correctly process the A1AT‐Z results in systemic deficiency leading to emphysema. The following study used cotransfection of mutant human A1At‐Z and ubiqtuin ligases into a variety of tissue culture cell lines. We identified three novel lectin‐like E3 ubiquitin ligases FBG1, FBG2, and FBG4 that participate in the clearance of A1AT‐Z. We demonstrate that both FBG1 and 2, but not FBG4, bind and pulldown A1AT‐Z. Only FBG1 and FBG2, are found in the liver, the location of A1AZ‐Z production. Furthermore, we show that soluble A1AT‐Z is degraded by the ubiquitin proteasome system and by autophagy while the insoluble form of A1AT‐Z is predominately degraded by autophagy. Overexpression of FBG1 and FBG2 decreases the half‐life of A1AT‐Z from 13 hours to 4 hours and 10 hours respectively. Finally, we show that FBG1 may increase A1AT‐Z secretion, suggesting that FBG1 may be used to treat both lung and liver disease in A1AT‐Z deficient patients.
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 Results

Yeast 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.

Conclusions

J‐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.
Pin1 is overexpressed in tamoxifen‐resistant breast cancer and facilitates ubiquitin‐mediated degradation of cyclin dependent kinase 10
Authors: Choi Hong Seok , Khanal Prem
Published: June 2021

The aim of this study was to elucidate the mechanisms by which downregulation of CDK10 expression confers resistance to tamoxifen in breast cancer. Here, we show that peptidyl‐prolyl isomerase Pin1 downregulates CDK10 protein as a result of its interaction with and ubiquitination of CDK10, thereby affecting CDK10‐dependent Raf‐1 phosphorylation (S338). Pin1−/− mouse embryonic fibroblasts (MEFs) show higher CDK10 expression than Pin1+/+ MEFs, whereas CDK10 protein was downregulated in the rescued Pin1−/− MEFs after reexpression of Pin1. Pin1 silencing in SKBR‐3 and MCF7 cells increased the CDK10 expression. In human tamoxifen‐resistant breast cancer and tamoxifen‐resistant MCF7 cells, immunohistochemical staining and immunoblotting analysis shows an inverse correlation between the expression of CDK10 and the degree of tamoxifen resistance. There was also a positive correlation between the high level of P‐Raf‐1 (Ser338) and Pin1 in human tamoxifen‐resistant breast cancer and tamoxifen‐resistant MCF7 (TAMR‐MCF7) cells. Importantly, 4‐OH tamoxifen (4‐OHT), when used in combination with overexpressed CDK10 or Raf‐1 inhibitor, increased cleaved PARP and DNA fragmentation to inhibit cologenic growth of MCF7 cells and Tamoxifen‐resistant MCF7 cells, respectively. Based on these findings, we suggest that the Pin1‐mediated CDK10 ubiquitination is a major regulator of tamoxifen‐resistant breast cancer cell growth and survival.
Nck1—an Adaptor Protein Modulating Expression of the Endoplasmic Reticulum (ER) Stress Sensor IRE1α
Authors: Li Hui , Larose Louise
Published: June 2021

The unfolded protein response (UPR) is a cellular mechanism to cope with ER stress induced by alteration in ER homeostasis. Recently, ER stress has been implicated in insulin resistance. In fact, hyperactivation of the IRE1α‐JNK branch of the UPR secondary to obesity has been reported to impair insulin signalling. Interestingly, we identified the adaptor protein Nck1 as an important player in potentiating this UPR signaling pathway in liver. In addition, we observed that depletion of Nck1 resulted in improved hepatic insulin signalling. Therefore, we hypothesized that depletion of Nck1 improved insulin signalling by reducing IRE1α signalling. Testing this hypothesis, we found that basal Akt activation was increased in a PI3K‐dependent manner by depleting Nck1 in HepG2 cells. This correlates with increased levels of tyrosine‐phosphorylated (pY) proteins. Meanwhile, depletion of Nck1 led to reduced IRE1α protein levels and signalling. By blocking Akt activation in Nck1 depleted cells, we further demonstrated that decreased IRE1α expression was independent on Akt activation. Finally, we observed that HepG2 cells depleted of IRE1α also displayed enhanced Akt activation and pY proteins levels. Overall, our data provide strong evidence showing that Nck1 in hepatic cells regulates Akt activation by modulating IRE1α expression and signalling. Supported by Canadian Institutes of Health Research.
14‐3‐3£m induces HSF‐1 and HSP70 expression via a GSK‐3£]/£]‐catenin‐dependent mechanism in hepatocellular carcinoma
Authors: LIU CHIACHIA , Jan Yee Jee , Sung Li Ying , Liou Jun Yang
Published: June 2021

14‐3‐3 proteins are a family of regulatory molecules which are highly conserved among all eukaryotic cells. 14‐3‐3 proteins composed of seven isoforms which play crucial roles in regulating multiple cellular processes Previous studies have indicated that several 14‐3‐3 isoforms are overexpressed and potentially play as the prognostic markers to predict the outcome of hepatocellular carcinoma (HCC). In this study, we demonstrate that 14‐3‐3σ is also overexpressed in HCC. We further investigate the potential downstream targets of 14‐3‐3σ in regulating HCC tumor progression. Results from analysis of gene expression profile revealed that 14‐3‐3σ regulates various tumor progression‐associated genes expression. We discovered that expression of heat shock protein (Hsp70) was increased in 14‐3‐3σ overexpressing HCC stable cells. Significantly correlation of 14‐3‐3σ and Hsp70 expression was confirmed in primary HCC tumors which were analyzed by immunohistochemistry. Furthermore, we found that 14‐3‐3σ‐induced Hsp70 expression was medicated through the activating and upregulating of heat shock factor‐1α (HSF‐1α). Additionally, 14‐3‐3σ enhanced HSF‐1α expression was significantly abolished by activation of GSK‐3β or knockdown of β‐catenin with siRNA. Conclusively, we propose a hypothesis that elevated 14‐3‐3σ expression may protect HCC survival by increasing HSF‐1/Hsp70 expression via a GSK‐3β/β‐catenin‐dependent mechanism.
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.
Millitarin‐1(M1), a synthesized isooctylphenyl derivative induced mitochondrial dependent apoptosis and ROS generation in A549 Cells
Authors: Kim Tae-Woong , Lim Mi-Hee , Yoon Deok-Hyo , Park Haeil , Cho Jae-Youl , Sung Gi-Ho , Lee Tae-Ho
Published: June 2021

Millitarin‐1(M1), a synthesized isooctylphenyl derivative on the basis of millitarin, which is found from Cordyceps millitaris. Cordyceps millitaris is a parasitic fungus and has long been used as a Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and anticancer. M1 suppresses effectively proliferation of A549, human lung adenocarcinoma, by causing apoptosis. M‐1 triggered the mitochondrial apoptotic pathway, as indicated by a change in proapoptotic moleculas(Bax, Bak, Bim)/ant‐apoptotic molecules(Bcl‐2, Bcl‐xL) ratios, resulting in cytochrome c release, and caspase‐3 and 9 activation. We also found that the generation of reactive oxygen species(ROS) is a critical mediator in the inhibition of M1 induced cell growth of A549. We observed that M1 treatment significantly inhibited cell growth in a concentration dependent manner and caused sub G0/G1 phase cell cycle arrest. Enhancement of ROS by M1 activated apoptosis and resulted in the increased activation of c‐Jun NH2‐terminal kinase, p38 and ERK1/2. Therefore, it indicated that M1 may be a potential candidate in future research and development of bioactive ingredients as anticancer agents. Supported by a grant from RDA(No. PJ008321), Korea
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
Strength differences between traditional medicinal preparations of cimicifuga racemosa, chamomilla rectita and petroselinum crispum
Authors: Rae Guenevere , Price Harry , Skelton Michele
Published: June 2021

The purpose of this study is to analyze the strength differences between traditional medicinal preparations (commonly referred to as a tincture and an infusion). One plant was chosen from each of the following categories depending the part of the plant used in traditional medicine: root used (Cimicifuga racemosa); flower used (Chamomilla rectita); and leaf used (Petroselinum crispum). Plants were obtained and cultivated in a controlled environment until harvesting. Published traditional medicine methods were utilized to prepare herbal preparations for both the infusion and the tincture process. High Performance Thin Layer Chromatography was used to separate chemical markers. The chemical markers (deoxyactein for Cimicifuga racemosa, and Apigenin for Chamomilla rectita and Patroselinum Crispum) were compared to standards and used to estimate strength differences between the preparations. Results show significant strength differences between the Cimicifuga racemosa preparations and no significant differences between the Chamomilla rectita and Petroselinum crispum preparations (p≤ 0.05). Results suggest that it may not be necessary to undergo the increased resource and time demand of the tincturing process in preparing leaf and flower traditional medicine preparations.
Transcriptional profiling of Yersinia pestis
Authors: Dimitrov George I , Gautam Aarti , Miller Stacy-Ann , Hoke Allison , Moyler Candace C , Jett Marti , Hammamieh Rasha , Nabarun Chakraborty
Published: June 2021

Yersinia pestis, the causative agent of plague, is known to have evolved a variety of mechanisms to overcome the host immune system and establish infections. The molecular changes induced by Y. pestis in the host are not well delineated. Here, we examine the events that occur on molecular and cellular levels after nonhuman primates are exposed to aerosolized CO92 Y. pestis. The transcriptional profile underwent drastic changes during the course of infection. In particular, we identified sets of genes at early and late time periods in blood, lungs and other organs. We discovered the HIF‐1 pathway that is altered in the infected monkeys, and have also characterized channels and transport mechanisms that are involved in the pathogenesis. Quantitative real‐time PCR was used to validate the microarray results. We describe profiles generated from a few genes that are involved in immune response, apoptosis, and molecular transport over the course of the infection.
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

Objective

We aimed to study whether oral administration with transgenic milk expressing a dust mite allergen can down‐regulate the allergen‐specific airway inflammation.

Methods

The 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.

Results

Upon 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.

Conclusion

In animal model, oral administration of transgenic milk expressing Dp2 allergen was demonstrated to have tolerance induction and protection from allergic airway inflammation.
Aerosolized Human Extracellular Superoxide Dismutase Prevents Hyperoxia‐Induced Lung Injury in Mouse Model
Authors: Chen Hsiao-Ling , Chen Chuan-Mu
Published: June 2021

Objective

An important issue in critical care medicine is the identification of ways to protect the lungs from oxygen toxicity and reduce systemic oxidative stress. The current study investigated the ability of aerosolized extracellular superoxide dismutase (EC‐SOD) to protect the lungs from hyperoxic injury.

Methods

Female CD‐1 mice were exposed in hyperoxia (FiO2 > 95%) to induce lung injury. The therapeutic effects of EC‐SOD via an aerosol delivery system for lung injury and systemic oxidative stress at 24, 48, 72 and 96 h of hyperoxia were measured by bronchoalveolar lavage, wet/dry ratio, histology, and 8‐oxo‐dG in lung tissue.

Results

After exposure to hyperoxia, the wet/dry weight ratio remained stable before day 2 but increased significantly after day 3. Treatment with aerosolized rhEC‐SOD increased the survival rate at day 3 under hyperoxia to 95.8%, which was significantly higher than that of the control group (57.1%) and albumin treated group (33.3%). The protective effects of EC‐SOD against hyperoxia were further confirmed by reduced lung edema and systemic oxidative stress.

Conclusion

The results encourage the use of an aerosol therapy with EC‐SOD in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure, including acute respiratory distress syndrome (ARDS).
Development of a high‐throughput screening approach targeting inhibitors of bacterial cell wall synthesis
Authors: White Courtney L , Gober James W
Published: June 2021

Many current antimicrobials act upon the synthesis of the peptidoglycan (PG) cell wall. The proteins that synthesize, assemble, and degrade PG are critical for cell viability. However, evolving antibiotic resistance places a new urgency in prokaryotic biology for new targets. In rod‐shaped bacteria, a large complex of protein interactions coordinate PG synthesis. At the heart of this complex exists an association recently found between two well‐conserved proteins ‐ an actin homolog (MreB) and a bitopic membrane protein (RodZ), both vital for cell wall maintenance. The interaction is confirmed in several species by various methods, one including a bacterial two‐hybrid system. We have adapted this system to high‐throughput screening (HTS) to search for inhibitors of the Caulobacter crescentus MreB‐RodZ interaction. This 384‐ well adaptation involves several original protocol modifications for final assay validation. With this assay, we have screened more than 70,000 unique compounds, producing 485 potential hits. A secondary counter‐screen and confirmatory experiments determine hits specific to RodZ‐MreB interaction. This work demonstrates two‐hybrid HTS assay feasibility and may potentially provide novel inhibitors of these proteins. It also may prove useful for testing of other essential protein interactions. This work is funded by a NIH Exploratory Research Grant.
The effect of bile acids on mitochondrial mass and activity in normal and cancer cells
Authors: Narendran Nirushan Harushikesh , Dvorak Katerina
Published: June 2021

Barrett's esophagus is a condition where normal squamous epithelium is replaced by metaplastic columnar epithelium. This condition is associated with an increased risk for the development of esophageal adenocarcinoma, which is linked to the reflux of bile acids. Our study was to compare the effect of bile acids (deoxycholic acid, DCA) on cytotoxicity, mitochondrial mass and activity in esophageal adenocarcinoma cells (JHEsoAd) and non dysplastic Barret's esophagus (CP‐A). Cytotoxicity was examined by SRB colorimetric assays, the activity of mitochondrial dehydrogenases was evaluated by MTS assay and mitochondrial mass was examined by MitoTracker Green and Hoechst staining. SRB showed a decrease in protein concentration in CP‐A cells exposed to DCA compared to untreated cells. A similar decrease was observed in JHEsoAd cells exposed to DCA. MTS showed a decrease in mitochondrial activity in CP‐A cells exposed to DCA but, the opposite effect was detected in JHEsoAd cells exposed to DCA. The ratio of the number of mitochondria (MitoTracker Green) and the number of cells (Hoechst) was observed. This ratio was increased in JHEsoAd1 cells but not in CP‐A cells. This indicated that the mitochondria in cancer cells respond differently to stress induced by bile acids and are crucial for apoptosis induction. Therefore, we will further evaluate the role of mitochondria in bile‐acid induced cancer development.
Influence of Epigenetic Histone Modifiers on PTEN/PI3K/Akt Signaling in Vascular Smooth Muscle Cells (VSMC)
Authors: Ranganna Kasturi , Mathew Omana , Milton Shirlette , Yatsu Frank
Published: June 2021

Activation and proliferation of VSMC are principal contributors to vascular remodeling and restenosis. Evidence indicates PTEN/PI3K/Akt signaling is important in the regulation of VSMC proliferation and contractility. Several epigenetic histone modifiers with HDAC inhibitory activity arrest VSMC proliferation and alter gene expression implicating involvement of epigenetic mechanism in vascular remodeling. Accordingly, butyrate and trichostatin A (TSA), well‐studied histone modifiers, arrest VSMC proliferation and alter cell cycle proteins expression. Since PTEN/PI3K/Akt signaling is important in the regulation of VSMC proliferation, here we investigate whether butyrate and TSA alter PTEN/PI3K/Akt signaling fittingly to cause arrest of VSMC proliferation. Proliferating VSMC treated with butyrate or TSA for different lengths of time were used to determine the activation state and expression levels of signaling proteins by western blotting. Analysis of phosphorylation states of PTEN, Akt and PDK1 reveals time‐dependent inhibition of Akt/PKB and PDK1 phosphorylation and stimulation of PTEN phosphorylation. Regarding downstream effectors, while phosphorylation of FOX3 is inhibited, no change in GSK3α/β phosphorylation state was observed in treated VSMC in addition to downregulation of NF‐KappaB. Taken together, it appears that by dampening several actions of Akt that are linked to its pro‐cell proliferation effects, butyrate and TSA cause arrest of VSMC proliferation SUPPORT: G12RR003045‐21 and C06RR012537 from NIH/NCRR
Oxidized lipids and lysophosphatidylcholine (LPC) induce the chemotaxis and intracellular calcium influx in human natural killer (NK) cells
Authors: Rolin Johannes , Al-Jaderi Zaidoon , Maghazachi Azzam A
Published: June 2021

We previously reported that human NK cells express G2A and they respond to LPC. Here we report that oxidized lipids such as 9‐ R‐HODE, 9‐S‐HODE and 13‐R‐HODE, as well as LPC induced the in vitro chemotaxis of human NK cells, although with variable efficacies. The chemotactic effects of these lipids were inhibited by prior treatment of NK cells with pertussis toxin. Further, anti‐G2A inhibited the chemotactic effects of all four lipids examined. 9‐S‐HODE, 9‐R‐HODE and LPC optimally induced the influx of intracellular Ca2+ in NK cells. Addition of 9‐S‐HODE prior to the addition of LPC inhibited more than 50% of the effect of LPC, whereas addition of LPC prior to the addition of 9‐S‐HODE completely inhibited the effect of the latter lipid. However, there was a complete reciprocal desensitization among 9‐R‐HODE and LPC on the influx of intracellular Ca2+. Further analysis showed that 1 μM concentration of all four lipids inhibited NK cell lysis of tumor target cells. These lipids did not affect the expression of chemokine receptors on NK cells. In contrast, we observed increased expression of CCR7, CCR9, CXCR1 and CXCR4 (9S‐HODE also increased expression of CCR6), but not CCR1, CCR2, CCR3, CCR4, CCR5, CXCR3, CXCR5 or CXCR6 after incubation of monocytes with these lipids. This was corroborated with the ability of lipid‐treated monocytes to respond chemotactically towards MIP‐3β/CCL21 and IL‐8/CXCL8, albeit with variable efficacies. In conclusion, we provide evidence showing that oxidized lipids and LPC exert important functions for cells of innate immune system.
The ubiquitin‐like (UBX)‐domain‐containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis
Authors: Wang Chao-Wen
Published: June 2021

Lipid droplets (LDs) are central organelles for maintaining lipid homeostasis. However, how cells control the size and number of LDs remains largely unknown. Herein, we report that Ubx2, a UBX‐domain‐containing protein involved in endoplasmic reticulum (ER)‐associated degradation (ERAD), is crucial for LD maintenance. Ubx2 redistributes from ER to LDs when LDs start to form and enlarge during diauxic shift and in the stationary phase. ubx2Δ cells contain abnormal number and reduced size of LDs and their triacylglycerol (TAG) is reduced to 50% of the normal level. Deletion of either UBX or UBA domain in Ubx2 has no effect, but deletion of both causes LD phenotypes similar to that in ubx2Δ. The reduced TAG in ubx2Δ is likely due to mislocalization of Lro1, one of the two TAG‐synthesizing enzymes in yeast, which moves along the ER and distributes dynamically to the putative LD assembly sites abutting LDs. Thus, Ubx2 is important for the maintenance of cellular TAG homeostasis likely through Lro1. The mammalian Ubxd8 expressed in yeast complements the defect of ubx2Δ, implying a functional conservation for these UBX‐domain‐containing proteins in lipid homeostasis.
The role of microtubule on dithiocarbamate cytotoxicity
Authors: Gonzalez-lopez Eugene , Cheng Shu-Yuan
Published: June 2021

Maneb (MB), mancozeb (MZ) and diethyldithiocarbamate (DDC) are fungicides widely used in the United States. These compounds can potentiate the presence of the dopaminergic neurotoxin (MPTP). However, the mechanisms for those effects are not so clear. Dopamine transporter (DAT) is essential for MPTP toxicity by transporting MPP+, the active metabolite of MPTP, into dopaminergic neurons. Preliminary results show that MB and MZ increase the interaction of DAT and alpha‐synuclein followed by accumulating cell surface DAT expression which in turn enhances MPP+ cytotoxicity by increasing MPP+ uptake. The disruption of the interaction of alpha‐synuclein with microtubules has been showed to enhance cell surface recruitment of DAT. The aim of this study is to elucidate the role of microtubules on dithiocarbamate cytotoxicity. Immunocytochemistry for the microtubule network was performed on PC12 cells treated with DDC (75 uM), MB (20 uM) and MZ (20 uM) for 4 hours. The images showed that those dithiocarbamates disturbed the cytoskeleton network, with MZ having shown the most severe depolymerization. Cells pre‐treated with microtubular stabilizer Taxol showed a significant alleviation in cell damage assayed by cell viability MTT assay. These results indicate that these compounds directly influence the microtubules; however, dithiocarbamates might also trigger other toxic mechanism resulting in cell death.
Isolation of secreted glycoproteins using sugar‐azide labeling and alkyne‐bead capture
Authors: Drake Richard R , Roper Stephen , Jones Ellen , Kraft Andrew S , Zemskova Marina
Published: June 2021

Proteomic methods used to characterize proteins present in the secretome of cultured cells frequently identify many non‐glycosylated cytosolic and cytoskeletal proteins. To address this, metabolic labeling was done in a prostate stroma cell line YPMI‐1 with an azide‐sugar analog of sialic acid, N‐acetylamannosamineazide (ManNAcAz), that is incorporated into secreted glycoproteins. Serum‐free media from cells with and without ManNAcAz labeling was collected after 24 hrs. The ManNAcAz‐treated media was incubated with alkyne beads and copper catalyst for crosslinking to glycoproteins. Bead‐bound proteins and conditioned secretome media were digested with trypsin, and proteins identified using electrospray tandem mass spectrometry. Over 400 proteins were detected in the secretome media, while 75 proteins were captured in ManNAcAz‐labeled media. The majority (>80%) of the identified proteins in the secretome media were cytosolic or cytoskeletal in origin and non‐glycosylated, while >90% of the ManNAcAz proteins were associated with secretion, extracellular matrix, adhesion or ligand/receptor functions. One protein detected was TGF‐beta2, which by ELISA was determined to be present at 5–10 ng/ml. This illustrates the depth and selectivity of detection provided by the azide‐alkyne capture method, and the method can readily be applied to many cell systems.