bluuebunny/crossref_metadata_2025 huggingface dataset:
Endosomal sorting of VAMP3 is regulated by Phosphatidylinositol 4‐kinase IIa
Authors: Jovic Marko , Kean Michelle , Gingras Anne‐Claude , Brill Julie , Balla Tamas
Published: June 2021

Membrane fusion along endocytic pathways is directly dependent on proper distribution of Soluble NSF‐attachment receptors (SNAREs). Although SNAREs are fairly promiscuous in vitro, significant specificity is achieved in vivo through spatial segregation and shielding of SNAREs prior to cognate Q‐SNARE binding. In particular, long R‐SNAREs are capable of autoinhibition, while short R‐SNAREs were shown to associate with phospholipids on vesicular membranes in vitro. However the exact regulation of this process and the potential lipid binding preference in vivo remains elusive.

Here we have identified PtdIns4P kinase PI4KIIa as a binding partner of VAMP3, a small recycling R‐SNARE. PI4KIIa depletion resulted in accumulation of VAMP3 on endosomal compartments, inhibited VAMP3 trafficking to perinuclear membranes and reduced the rate of VAMP3‐mediated transferrin recycling. Similarly, acute depletion of PtdIns4P on endosomes significantly impaired VAMP3 sorting. In addition, VAMP3 association with its cognate Q‐SNARE Vti1a decreased upon PI4KIIa depletion.

Taken together, these findings reveal a role for phospholipids in small R‐SNARE sorting, corroborating previous in vitro findings. Direct interaction with the lipid kinase PI4KIIa can, therefore, guarantee a localized supply of PtdIns4P to support the lipid requirement of endosomal SNARE function.

Supported by the NICHD Intramural program
Real Time Trafficking of System xc− in COS‐7 cells
Authors: Cramer Cassondra , Chase Leah
Published: June 2021

The cystine/glutamate exchanger, System xc−, is comprised of two proteins, xCT and CD98 and is known to play a significant role in the protective response of cells to oxidative insult. We have previously shown that System xc− activity is rapidly upregulated in the presence of oxidative agents as a result of an increase in trafficking of xCT to the plasma membrane, without an equivalent increase in CD98 membrane localization. Since CD98 has been suggested to be involved in the trafficking xCT, we sought out to monitor the association between xCT and CD98 during this trafficking event. To address this question, we have created three constructs: xCT‐GFP, GFP‐xCT, and mCherry‐CD98 and expressed them in COS‐7 cells. Addition of the GFP tag to either the N‐terminus or the C‐terminus of xCT does not disrupt the basal trafficking of xCT in COS‐7 cells, as 34% of the GFP tagged xCT proteins are localized to the membrane under basal conditions, similar to endogenously expressed xCT. Using live cell fluorescence microscopy, we have observed that while some of GFP‐xCT (or xCT‐GFP) is co‐localized with mCherry‐CD98 on the membrane and within vesicular pools within the cytoplasm, distinct pools of intracellular mCherry‐CD98 and GFP‐xCT do not co‐localize. We are currently monitoring the real time trafficking of xCT‐GFP and mCherry‐CD98 in response to oxidant exposure. This research is supported by NSF‐RUI # 0843564.
The Role of Endosomal Phosphoinositides in Phagocytosis, Phagosome Maturation and the Endosomal System in Macrophages
Authors: Kim Grace Ha Eun , Dayam Monica , Botelho Roberto
Published: June 2021

As part of an innate immune response, macrophages engulf pathogens into phagosomes for degradation through a process known as phagocytosis. These newly formed phagosomes progressively mature and fuse with early and late endosomes and lysosomes to form phagolysosomes, where pathogens are degraded by hydrolytic enzymes. Phosphatidylinositol‐3‐phosphate [PtdIns(3)P] and phosphatidylinositol‐3,5‐bisphosphate [PtdIns(3,5)P2] are signaling lipids that recruit a unique set of effector proteins involved in distinct stages of membrane traffic to govern the function of early and late endosomes, respectively. Phagosome maturation involves the transient expression of PtdIns(3)P on early phagosomal membranes. Subsequently, PtdIns(3)P can be converted to PtdIns(3,5)P2 by the lipid kinase PIKfyve. In this study, a pharmacological approach is employed to dissect the individual roles of PtdIns(3)P and PtdIns(3,5)P2 in phagocytosis, phagosome maturation and the endosomal system. Our immunofluorescence data show that RAW264.7 macrophages treated with MF4, a selective inhibitor of PIKfyve, display a moderate defect in endocytosis and phagosomal acquisition of lysosomal‐associated membrane protein 1 (LAMP‐1), while phagocytosis and the degradative capacity of lysosomes remain unperturbed. Funded by Ryerson University and NSERC.
Cholesteryl ester transfer protein deficiency in adipocytes disrupts triglyceride metabolism in multiple ways
Authors: Greene Diane Jackel , Morton Richard E
Published: June 2021

Previous studies implicate cholesteryl ester transfer protein (CETP) in the intracellular transport of triglyceride (TG) from the endoplasmic reticulum (ER) to lipid storage droplets (LD). Here, we further characterize TG metabolism in modified SW872 adipocytes expressing only 25% of normal CETP. Measured by oleate incorporation, TG synthesis in CETP deficient cells was 75% of control but in vivo transport of this newly synthesized TG from the ER to LD was only 40% of control, correlating with the reduced ability of these cells to accumulate TG in LD. Despite reduced transfer, the TG content of cellular membranes was not increased. During TG biosynthesis, CETP deficient cells accumulated a high level of diglyceride (DG), a pathway intermediate. Although TG synthesis was lower, TG + DG synthesis in CETP deficient cells was 140% of control. Consistent with this, mRNA and protein levels of glycerol 3‐phosphate acyltransferase were elevated. DG acyltransferase 1 and 2 mRNA levels were unchanged but adipocyte TG lipase mRNA was increased 2.2 fold. The mismatch between rates of TG synthesis and storage in CETP deficient adipocytes may result from the failure to translocate TG from the ER to LD, subsequently impairing DGAT activity and/or inducing TG hydrolysis in the ER, causing a rise in DG. Intracellular CETP appears to be necessary for efficient synthesis of storage lipids and their targeting to lipid droplets. NIH HL060934
Both full‐length and exon 9‐deleted human cholesteryl ester transfer protein isoforms are involved in cellular lipid homeostasis
Authors: Izem Lahoucine , Morton Richard E
Published: June 2021

In humans, cholesteryl ester transfer protein (CETP) exists as full‐length (FL) and exon 9‐deleted (E9) isoforms. The E9 variant is retained by cells while FL CETP is largely secreted. We reported before that simultaneously inhibiting both FL and E9 CETP synthesis in SW872 human adipocytes disrupts their lipid metabolism. To further investigate the roles of these two isoforms in cellular lipid metabolism, we stably expressed each isoform in human cells that naturally have CETP and mouse cells that lack CETP. The expression of FL‐CETP in mouse 3T3‐L1 cells increased their droplet cholesteryl ester (CE) content by 2 fold and their lipid efflux capacity by more than 20%. Expressing E9 CETP increased CE and triglyceride (TG) by 17% and 30% respectively. In human SW872 cells overexpressing FL‐CETP, lipid efflux was increased 2‐fold compared to control, but they stored 50% less TG in smaller, more numerous lipid storage droplets. Overexpression of E9‐CETP reduced the amount of secreted FL‐CETP by 75% and increased their CE and TG content by 2 fold, which can be partly explained by a significant reduction in lipid efflux and a slight increase in TG synthesis. In vitro, while E9 CETP blocks completely the FL‐CETP mediated transfer of lipids between organelles, E9 CETP showed some lipid transfer activity on its own. Collectively, our data implicate both FL‐length and E9 CETP in modulating cellular lipid metabolism. NIH HL 060934
Characterization of a perilipin 5 splice variant
Authors: Hubbell Theresa C. , DuBreuil Daniel M. , Tansey John T.
Published: June 2021

The perilipins are a family of five related genes that code for proteins which play seminal roles in lipid storage. At least one of these proteins (perilipin 1) has been shown to have several truncated forms that arise through differential mRNA splicing. Using western blotting we have identified a truncated form of perilipin 5 we have termed perilipin 5 b. This protein is found in murine heart tissue as well as cultured cells. We hypothesized that this was a novel splice variant of the perilipin 5 mRNA. Using RT‐PCR we have identified multiple amplicons suggesting that perilipin‐5b contains a read through of intron 8 of the murine perilipin 5 gene. shRNA studies conducted in cultured cells result in decreased levels of the protein. In addition, immunofluorescence microscopy revealed that perilipin‐5B is unable to bind to mature lipid droplets, but does form distinct puncta in the cytosol. Taken together, this data suggests that perilipin‐5B is a splice variant of perilipin‐5 found in oxidative tissues that is unable to bind to lipid droplets. The role of this protein in neutral lipid metabolism remains unclear.
Acrolein, a component of tobacco smoke and age‐related oxidative stress disrupts structure and function of Apolipoprotein E
Authors: Tran Tuyen Ngoc , Lee Yuan Yu , Uchida Koji , Narayanaswami Vasanthy
Published: June 2021

Apolipoprotein E (apoE), an anti‐atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for low‐density lipoprotein receptors (LDLr). ApoE mediates this function via essential lysine residues that interact with the LDLr. The objective of this project is to study the effect of oxidative stress (specifically acrolein) mediated in vitro modification on the structure and function of recombinant rat apoE. SDS‐PAGE and RP‐HPLC confirmed that the protein was purified to homogeneity with no signs of degradation. Acrolein modification of apoE was confirmed by Western blot. Guanidine HCl‐induced denaturation revealed that the overall fold of the modified protein was significantly altered. Modified apoE also demonstrated a decrease in heparin binding affinity and lipid binding ability. The LDLr binding ability of modified apoE was significantly impaired. Lastly, MALDI‐TOF/TOF MS analysis identified Lys82, 85, 86, 156 and 252 as likely modification sites by acrolein. Overall, we conclude that acrolein disrupts structural and functional integrity of apoE, which is likely to affect its role in maintaining plasma cholesterol homeostasis.
A characterization of the modular nature of histone‐derived antimicrobial peptides
Authors: Bustillo Maria E , LaBouyer Maria A , Fischer Alexandra L , Webb Drew C , Elmore Donald E
Published: June 2021

We have investigated the bactericidal activity, specificity, and mechanism of a several antimicrobial peptides (AMPs) derived from histone H2A alone and combined into chimeric peptides. Relative antibacterial efficacy of these AMPs was determined using radial diffusion assays, and eukaryotic cytotoxicity assays indicate that these peptides demonstrate bacterial specificity. Confocal microscopy was used to determine the mechanism of action of the peptides. Parasin, buforin II, and buforin I are segments of the larger hipposin AMP. Buforin II can translocate membranes while parasin is a permeabilizing peptide. Buforin I and hipposin are larger peptides containing both parasin and buforin II segments, and both are permeabilizing peptides relatively unable to translocate the membrane. These findings suggest that the addition of a lytic fragment to an otherwise‐translocating peptide confers a lytic property. We further explored this hypothesis by studying a chimeric peptide of parasin and DesHDAP1, another peptide known to translocate effectively. This chimera was found to be more strongly bactericidal than either parasin or DesHDAP1 alone.
Biochemical analysis unveils the mechanism of the interaction between Hsp70s and lipids
Authors: McCallister Chelsea , Nikolaidis Nikolas
Published: June 2021

Hsp70s are molecular chaperones essential for cellular homeostasis and survival. Several Hsp70s associate with cellular membranes, where they bind to specific lipids. The association of Hsp70s with lipids anchors them to the plasma membrane, where they regulate cell signaling, stabilizes the lysosomal membrane, and is required for microautophagy. Despite these essential functions the Hsp70‐ lipid interactions remain largely uncharacterized. To understand the molecular mechanism of these interactions we characterized the binding of HSPA1A, a cytosolic hsp70, to lipids. We determined the binding affinities of recombinant HSPA1A to several negatively charged phospholipids and established that although lipid‐binding is mediated largely by the nucleotide‐binding domain, allosteric communication between the two protein domains is crucial for lipid‐binding. We also determined that a conformational change caused by nucleotide‐binding reduces lipid‐binding, while binding of protein substrates has no effect on the lipid‐binding. Next, we determined that the HSPA1A‐lipid interaction is not purely electrostatic and the protein is most probably embedded in the lipid‐bilayer. Finally, we observed that binding of HSPA1A to lipids alters the refolding activity of the chaperone, which suggests that lipid‐binding could represent a new regulatory mechanism of chaperone function.

This project was supported by funds from CSUPERB and CSUF to NN
Structural insights into the role of BamA in the biogenesis of beta‐barrel membrane proteins in Gram‐negative bacteria
Authors: Noinaj Nicholas , Kuszak Adam , Gumbart JC , Lukacik Petra , Chang Hoshing , Easley Nicole , Lithgow Trevor , Buchanan Susan K
Published: June 2021

The mechanism for integration of α‐helical membrane proteins into membranes has been well established. However, the mechanism for insertion of β‐barrel membrane proteins, a unique type of membrane protein that can only be found in the outer membranes of Gram‐negative bacteria, mitochondria, and chloroplasts, remains elusive. For Gram‐negative bacteria, a five‐component complex called the β‐barrel assembly machinery (BAM) complex is required for membrane integration. To understand the mechanism of the BAM complex, we have determined crystal structures of BamA, a β‐barrel membrane protein itself, in two different conformations representing open and closed states. The structure of BamA contains a C‐terminal β‐barrel domain consisting of 16‐strands and a large periplasmic domain which sits in close proximity to the periplasmic face of the β‐barrel domain in the closed state. Our studies suggest that BamA may function by priming the local membrane for insertion and may even undergo a lateral opening event during protein insertion. This work is supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 20892.
Cell cycle regulation mechanism in pathogenic yeast, Cryptococcus neoformans: Structure‐function relationship of G1 and G1/S cyclins homologue CnCln1
Authors: Kawamoto Susumu , Virtudazo Eric V , Suganami Akiko , Tamura Yutaka
Published: June 2021

Cryptococcus neoformans is an opportunistic pathogen of worldwide distribution and responsible for life‐threatening infections among immunecompromised persons. We have reported that the cell cycle behavior of this yeast is different from the cell cycle control model exhibited by the model yeast Saccharomyces cerevisiae. The molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans Cyclin‐dependent kinase 1 (CnCdk1) and its G1 cyclin, in the pathogenic yeast, will be reported and discussed. Only a single Cdk1‐related G1 and G1/S cyclin homologue was found in the genome sequence of C. neoformans and designated it CnCln1. Surprisingly, CnCln1 was not only able to complement the function of the G1 cyclins of S. cerevisiae, such as ScCln3, but also the G1/S cyclins of S. cerevisiae, such as ScCln1 and ScCln2. Our in silico analysis demonstrated that the CnCln1/ScCdk1 complex was more stable than any of the yeast cyclin and ScCdk1 complexes. Thus, these results are consistent with in vitro analysis that has revealed the flexible functional capacity of CnCln1 as a Cdk1‐related G1 and G1/S cyclins of S. cerevisiae. This work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
Video time lapse imaging of mitotic catastrophe events in response to thiopurines
Authors: Patel Shikha , Pittman Douglas
Published: June 2021

Thiopurines are a class of chemotherapy drugs used as immunosuppressants in organ transplant patients and for the treatment of childhood cancers. However, approximately 40% of patients develop life‐threatening problems later in life as a direct result of treatment. 6‐thioguanine (6TG) is a thiopurine directly incorporated into the DNA. Processing of the damage by the mismatch DNA repair pathway leads to the formation of double‐stranded breaks repaired by the homologous recombination (HR) machinery. RAD51D is an HR protein, and absence of RAD51D leads to the formation of large multinucleated cells in response to 6TG treatment. Using live cell imaging, I am attempting to determine how these multinucleated cells form. Treated and untreated mouse embryo fibroblasts (MEFs) were cultured in a 37°C chamber containing 5% CO2 and visualized using a Leica ASMDW microscope. Images were captured every three minutes and translated into individual videos. Cell division times and outcomes were determined by manually following each cell and daughter cells. The data suggest multinucleated cells form after aberrant mitotic events and subsequent fusion of the daughter cells during the second cell cycle following treatment. Current data also indicate that multinucleation is more prevalent at lower doses while interphase arrest is more common at a higher dose of 6TG. These studies have the potential to determine the mechanisms of thiopurine‐induced secondary cancers and may possibly be used to identify individuals who may benefit from alternative treatments.
Identification and Characterization of Mitotic Defect‐related Proteins (MDr1 and MDr2) Necessary for Bipolar Spindle Assembly
Authors: Cheung Keith , Torres Jorge
Published: June 2021

During mitosis chromosome segregation relies on the proper formation and stabilization of the mitotic spindle. Although many proteins have been identified that contribute to spindle formation, presumably many others are involved. Therefore, we sought to identify novel proteins involved in mitotic spindle formation. To this end, mitotic HeLa cell extracts were used for in vitro microtubule polymerization reactions and proteins co‐pelleting with microtubules were identified by mass spectrometry. We generated a Dharmacon siRNA library directed against 600 microtubule binding proteins for use in various high throughput genetic screens, including mitotic spindle assembly, mitotic arrest, mitotic bypass, and apoptosis. Among the positive hits from these screens were a 55‐kDa protein MDr1 and a 42‐kDa protein MDr2. To characterize these proteins, I am taking both cellular and biochemical approaches. I have developed inducible GFP‐tagged MDr1 and MDr2 protein cell lines and show that MDr1 and MDr2 only co‐localize during mitosis. I also showed that recombinant MDr1 and MDr2 form a stable complex by in‐vitro binding experiments. The results suggested that MDr1 and MDr2 are regulated and only form a functional complex when entering mitosis.
Paradoxical Effects of Metformin on the Cell Cycle in L6 Myoblasts
Authors: Parakhia Rahul , Ochs Raymond S.
Published: June 2021

Metformin, the most commonly prescribed treatment for type II diabetes, is also known as an anti‐cancer agent. While most metabolic actions of metformin appear to act through AMP kinase (AMPK) there is currently a controversy about whether AMPK is involved in the long‐term actions of metformin. Recent reports have shown that cancer stem cells are preferentially inhibited by metformin, and shifted into a stationary growth cell phase. We found that L6 cell myoblasts also showed attenuation of growth during chronic treatment with 1 mM metformin. However, flow cytometry demonstrated that the surviving cells were paradoxically shifted into the S‐phase. These effects were reversed by the simultaneous presence of the AMPK inhibitor Compound C. By contrast, mouse embryonic stem cells exposed to 10 mM metformin for 24 h showed no shift in their cell cycle. These unexpected findings in comparison to the cancer cells may shed light on the mechanisms of normal development, embryonic development, and cancer in response to metformin.
Cranberry proanthocyanidins affect human prostate cancer cell growth via cell cycle arrest by modulating expression of cell cycle regulators
Authors: Hurta Robert , Kim Joseph , McKeown Brendan , Patel Kunal , Neto Catherine
Published: June 2021

The effects of a proanthocyanidin‐enriched fraction (PACs) from American cranberry(V. macrocarpon) on DU145 cancer cells’ behavior in vitro was studied. PACs was characterized by MALDI‐TOF MS to contain PAC‐oligomers ranging in size from 2–12 epicatechin units with at least one A type linkage. PACs (25 ug/mL) decreased cellular viability by ~30 % post 6h.of treatment. PACs (post 6h. exposure) increased the proportion of cells in G2‐M and decreased the proportion of cells in G1. These alterations in cell cycle were associated with changes in cell cycle regulatory proteins and other cell cycle associated activities. PACs decreased the protein expression levels of cyclin A & cyclin B1 and increased the expression of cyclin D1 & cyclin E. PACs treatment resulted in an increase in the protein expression levels of CDK2 & CDK4. Decreased p21, p16, & pRBp107 protein expression levels and increased p27 protein expression levels were evident in response to PACs treatment with no apparent alteration in the levels of pRbp130 protein. These findings demonstrate that PACs contribute to the cranberry mediated alterations in cell cycle proteins. Cranberry extracts can affect the behavior of DU145 prostate cancer cells in vitro further supporting the potential health benefits associated with cranberries. [N.C.I.C.‐ CCS, P.E‐HRP, The Cranberry Institute (Wisconsin Cranberry Board), Telus MRFD Prostate Cancer Fund (PEI Division)]
Effects of sulforaphane on three tumor cell lines and one normal cell line.
Authors: Jin Fang , Terwilliger Toby , Bosch Jessica , O'Donnell Robert W.
Published: June 2021

Sulforaphane (SFN) is an isothiocyanate found largely in cruciferous vegetables, which has been shown to have anti‐cancer effects, including the induction of apoptosis and cell cycle arrest. We tested the effects of SFN on three different cancer cell lines: HTB‐4, derived from a bladder cancer, MDA‐MB‐435, likely derived from a melanoma, and HL‐60, derived from a promyelocytic leukemia and one normal fibroblast cell line, MRC‐5. We found different abilities of SFN to induce apoptosis and arrest the cell cycle for the three cancer cell lines. HL‐60 cells were most sensitive to the SFN treatment, followed by HTB‐4 cells, while MDA‐MB‐435 cells were the least affected by SFN. Our results for the MRC‐5 cells showed that SFN was less toxic to them compared to the other three cancer cell lines. To investigate whether the drug resistance could be induced to SFN, we are testing HL‐60 cells that are exposed to SFN at low doses to see how easily resistance is generated but that has proven to be challenging and something we continue to work on. We conclude that the different sensitivities of these four cell lines to SFN are consistent with the heterogeneity of different cancers and may indicate an increased sensitivity of tumor cells to SFN. If we are unable to development SFN resistance in HL‐60 or any of the other tumor cell lines it would have better therapeutic potential, especially if normal cells are more resistant.
Pentoxifylline Initiates GSK‐3β‐Induced Proteasomal Degradation of Cyclin D1 and Arrests Renal Cancer Cells in the G1 Phase
Authors: Cai Wesley , Mastrandrea Nicholas J. , Tham Kim Y. , Monks Terrence J. , Lau Serrine S.
Published: June 2021

Cyclin D1 is required for cells to progress from G1 to S phase of the cell cycle. Mutation or over‐expression of cyclin D1 leads to uncontrolled proliferation. QTRRE (rat) and ACHN (human) renal cancer cells possess elevated levels of cyclin D1. Pentoxifylline (PTX), a non‐specific phosphodiesterase inhibitor, induces a time‐(0–24 hr) and dose‐(35 μM‐3.5 mM) dependent decrease in cyclin D1 levels in both cell models. QTRRE and ACHN cells treated with PTX (24 hr) undergo cell cycle arrest in the G1 phase (170 & 140%, respectively). While RT‐PCR showed no change in cyclin D1 transcription after PTX treatment (0–24hr), a decrease of cyclin D1 t1/2 was observed, suggesting an increase in protein degradation. Furthermore, PTX‐induced decrease of cyclin D1 was abolished in the presence of a proteasome inhibitor (MG‐132, 10μM) as well as GSK‐3β inhibitors (LiCl, 20mM and VI, 10uM). MS proteomics analysis following cyclin D1 antibody pull‐down revealed that cyclin D1 associates with proteasomal subunit α type 5 and polyubiquitins B & C. The data suggest that PTX decreases cyclin D1 levels by stimulating GSK‐3β‐induced proteasomal degradation, which promotes G1 phase cell cycle arrest. Moreover, because our findings reveal a novel anti‐cancer chemotherapeutic property of PTX, the utility of PTX as an adjuvant therapy in the treatment of cancer should be further explored. (AstraZeneca Studentship, T32ES016652, P30ES006694)
YAP mediates crosstalk between the Hippo and PI(3)K–TOR pathways by suppressing PTEN via miR‐29
Authors: Tumaneng Karen Andres
Published: June 2021

Organ development is a complex process governed by the interplay of several signalling pathways that play critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear. Here we provide evidence that YAP activates the mammalian target of rapamycin (mTOR), a major regulator of cell growth. We have identified the tumour suppressor PTEN, an upstream negative regulator of mTOR, as a critical mediator of YAP in mTOR regulation. We demonstrate that YAP downregulates PTEN by inducing miR‐29 to inhibit PTEN translation. Last, we show that PI(3)K–mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia. Our studies reveal a functional link between Hippo and PI(3)K–mTOR, providing a molecular basis for the coordination of these two pathways in organ size regulation.
Identification of regulatory factors of the stationary phase sigma factor RpoS in Escherichia coli
Authors: Nguyen Kien , Peterson Celeste
Published: June 2021

The sigma factor RpoS is the master regulator of the general stress response that helps cells survive harsh environmental conditions by associating with the RNA polymerase and regulating gene expression. While numerous stresses are known to up‐regulate RpoS, many of the molecular steps leading from stresses to changes in RpoS levels remain unknown. To screen for the novel regulators, we used an RpoS’‐‘LacZ fusion. We transformed the reporter strains with an overexpression library, screening for plasmids that resulted in colonies with either more or less RpoS as observed on Xgal indicator plates. We verified that RpoS levels were altered in the presence of the plasmid with Western blots and quantified the effect on RpoS expression beta‐galactosidase assays in both RpoS transcription and translation lacZ fusions. We identified the genes on the plasmids as gadY, rbsD, yhhI, yhjR, ykgR and part of the dsdA gene in the screen. Our results suggested all of the factors, with the exception of rbsD, regulated RpoS in a post‐transcriptional manner either at the level of translation or protein stability. The small RNA gadY caused a strong inhibitory effect on RpoS expression and yhhI, ykgR and yhjR causing a mild increase expression of RpoS. In summary, we have found multiple new factors that affect RpoS at the post‐transcriptional level.
Analysis of novel interacting protein partners of Wsc1p and Mid2p, two sensor proteins of the PKC1 signaling pathway
Authors: Santiago Ednalise , De Jesús Camille , Borrero Alexander , Caballero Natalia M. , Diaz Orlando J. , Stagljar Igor , Rodríguez‐Medina José R.
Published: June 2021

Wsc1 and Mid2 are protein sensors of cell wall stress that activate the PKC1‐Cell Wall Integrity Pathway in response to environmental stress. We hypothesized that Wsc1p and Mid2p attract additional interacting protein partners (interactors) that can be associated with PKC1 activation. To identify novel interactors, the integrated membrane Y2H (iMYTH) technique was applied. A cDNA plasmid library of prey proteins was expressed in bait strains containing iMYTH reporter modules of Wsc1p and Mid2p. The interacting prey plasmid clones were recovered and sequenced. To assess their functionality in PKC1 activation, we performed Western blot analysis of phospho‐Slt2p in null mutants of the interactors. Under normal culture conditions at 30°C, 14 novel interactors were confirmed for Wsc1p and 29 for Mid2p. In a second iMYTH screen performed under heat stress conditions at 37°C, 4 novel interactors for Mid2p and 1 for Wsc1p were identified. At 37°C, YHR193C was the dominant interactor for Wsc1p and Mid2p. Null mutants of the interactors YOL109W, YHR135C, YPL199C exhibited normal PKC1 activation status at 30°C and 37°C while theYHR193C null mutant failed to activate PKC1 at 37°C. In conclusion, YHR193C is a protein interactor of Wsc1p and Mid2p required for activation of the PKC1 pathway. Supported by NCRR‐RCMI‐G12RR03051, MBRS‐RISE‐R25GM061838 & Univ. of Toronto.
Analysis of MILLIPLEX® MAP NF[kappa]B signaling using Luminex® xMAP® technology
Authors: Hwang Joseph , Warmke Tim , Wiese Rick
Published: June 2021

NFκB (nuclear factor kappa B) is a transcription factor that plays a crucial regulatory role in both physiological and pathological processes, such as immune responses, inflammation, proliferation, necrosis, apoptosis and tumorigenesis. The main NFκB canonical pathway is composed of its receptor, TNF‐R1/2, adaptor proteins (TRADD, RIP1, and TRAF2), kinases (IKKα/β/γ), inhibitors (IκBα) and the NFκB transcription factor. The NFκB pathway is coordinately regulated by phosphorylation and understanding the role of this pathway in normal physiological processes and in diseases requires the ability to simultaneously measure phosphorylation status of multiple protein targets. In recent years, Luminex® xMAP® bead‐based technology, have enabled the measurement of up to 50 proteins simultaneously. Using the NFκB multiplex panel, we observed increased levels of phosphorylated NFκB, IκB, IKKα, IKKβ, IKKγ and FADD in TNFα treated cells. In contrast, no increase in phosphorylation was observed in cells treated with various other stimuli, including anisomycin, staurosporine, hydrogen peroxide, FasL and CoCl2. Modifications in phosphorylation levels were confirmed by the Western blotting using the detection antibodies from the NFκB multiplex panel. The NFκB multiplex panel allowed for simultaneous detection of multiple analytes in a specific, sensitive, and reproducible manner.
Regulator of G protein signaling 6 (RGS6) mediates doxorubicin‐induced myocardial cell apoptosis and cardiomyopathy
Authors: Yang Jianqi , Maity Biswanath , Huang Jie , Gao Zhan , Stewart Adele , Weiss Robert M , Anderson Mark E , Fisher Rory A
Published: June 2021

Activation of p53 and myocardial apoptosis are crucial events responsible for Dox‐induced cardiomyopathy and heart failure. Recently we discovered a novel signaling role for RGS6 in mediating activation by Dox of the ATM/p53 apoptosis pathway via reactive oxygen species (ROS), which are also strongly implicated in myocardial apoptosis. Here, we investigated whether RGS6 was involved in the pathogenic response to Dox in heart. While Dox‐treated wild type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, and decreased survival five days following Dox administration, RGS6−/− mice were completely protected against these pathogenic responses. The activation of ATM/p53‐apoptosis signaling by Dox in ventricles of WT mice was completely absent in their RGS6−/− counterparts. Dox‐induced ROS generation was dramatically impaired in ventricles and isolated VCM from RGS6−/− mice, and RGS6 was required for Dox‐induced apoptosis in VCM, which was also found to be ROS‐dependent. Thus, RGS6 is a previously unrecognized, but essential, mediator of the pathogenic responses to Dox in heart where RGS6 promotes activation of apoptotic signaling pathways by modulating Dox‐induced ROS generation. RGS6 inhibition might represent a viable means to circumvent the cardiotoxic actions of Dox in human cancer patients. (AHA 11SDG7580008 and NIH CA161882, HL079031, HL62494, HL70250 and HL113001)
Spice Synthetic Cannabinoid Drugs: Lung and Adrenal Cell Models
Authors: Vanderpuye Oluseyi Adewale , Smith Toni , Walker Brandon , Gilbert Myrone , Okediji Olatunde
Published: June 2021

‘Spice’ refers to designer drugs consisting of plant materials spiked with different synthetic mimetics of delta tetrahydrocannabinol, a psychoactive compound in marijuana. The toxicology and bioactivity of many synthetic cannabinoids are unknown including Spice brands: Barely Legal, Brain Storm and Voodoo.

It was hypothesized that the ‘Spice’ brands Barely Legal, Brain Storm and Voodoo Child contain different compounds, overlap in biological activities with delta THC and affect cell physiology and morphology. This study sought possible biomarkers for the effects of ‘Spice’. Fluorescence microscopy detected cannabinoid receptor type 2 proteins but not type 1 receptors on A549 and SW‐13 cells. The complement system regulatory proteins CD46 and CD59 but not CD55 were detected on A549 and SW‐13 cells. Glycoconjugate profiles on these cells were characterized by lectin binding. Exposure to delta THC and Barely Legal Spice caused stronger Con A lectin binding to A549 lung cells than did exposure to Brainstorm Spice or control media. This study is the first description of the complement regulatory protein profile of adrenal gland cells and provided carbohydrate structure profiles of A549 and SW‐13 cells as detected by lectin binding. The results will facilitate more detailed analysis of the toxicology and bioactivities of spice.
Production of transgenic mice expressing human protein C
Authors: MIN Kwan‐Sik , NANJIDSUREN TSEVELMAA , Park Chae‐Won , Yun Sung‐Jo
Published: June 2021

Human protein C (hPC) is a regulator of homeostasis, suggesting its potential use as a therapy for many disease states. Protein C is a zymogen of a serine protease that is activated by thrombin. Protein C, also known as autoprothrombin ¢òA and blood coagulation factor ¢û¢õ, is a zymogenic (inactive) protein, the activated form of which plays an important role in regulating blood clotting, inflammation, cell death and maintaining the permeability of blood vessel walls in humans and other animals. hPC is a 62 KD disulfide‐linked heterodimer consisting of a 21 KD light chain and a 41 KD heavy chain which circulates as an inactive zymogen in plasma.

In this study, we focus on generation of hPC transgenic mice. hPC transgenic mice were produced by using micro‐injection method. The hPC cDNA was cloned into pBC1 vector under goat beta‐casein promoter. One‐cell stage embryos microinjected were transferred to 24 recipient mice on day 1 of the estrus cycle. We screened 61 mice by the PCR. Four line transgenic mice were identified and confirmed expression of protein C gene in mammary gland and several organ. We also analyzed the expression of mRNA and protein through the northern blot and western blot in mammary gland of hPC transgenic mice. hPC was localized in the alveolar epithelial cell by immunohistochemistry. Now, we are collecting the milk from the 2 found lines.
Mutation of Non‐Essential Cysteines Leads to Highly Soluble and Active Recombinant Full‐Length NEMO
Authors: Cote Shaun M , Herscovitch Melanie , Prenovitz Robert , Ennis Thomas , Gilmore Thomas D. , Whitty Adrian
Published: June 2021

NF‐κB essential modulator (NEMO) is a major component of the Iκ Kinase (IKK) complex, which serves as a critical component of NF‐κB signaling. In the IKK complex, NEMO forms a 2:2 interaction with its binding partner Iκ Kinase β (IKKβ). Small molecule inhibitors that specifically block the interaction of NEMO with IKKβ would have potential utility in inflammatory diseases and certain types of cancer. Generating a recombinant form of full‐length NEMO for biochemical characterization and inhibitor discovery has proven to be problematic, however, due to disulfide‐mediated misfolding and aggregation involving some of NEMO's 11 cysteine residues. We show here that highly soluble and homogeneous full‐length NEMO can be generated by mutating five or seven non‐critical cysteine residues. Both the 5xAla and 7xAla mutants show wild‐type affinity for IKKβ in a fluorescence anisotropy binding assay. Morevover, when transfected into NEMO −/− fibroblasts, both mutants were able to restore NF‐κB‐dependent cell survival in response to TNFα treatment. Our results show that none of the seven mutated cysteines are required for the IKKβ binding activity of NEMO, and identify a form of full‐length recombinant protein suitable for screening assays and structural characterization. (This work was supported by NIH GM094551 to AW and TDG)
Characterization of simvastatin‐induced structural and functional alterations in the molecules of kidney brush border membrane by ATR‐FTIR spectroscopy and chemometric approaches
Authors: Severcan Feride , Gocmen Semire Uzun , Ozek Nihal Simsek , Severcan Mete
Published: June 2021

Simvastatin, lipophilic statin, is the most commonly used drugs to treat hyperlipidaemia. Recent studies reported that they have both adverse and beneficial effects on kidney. However these results are contradictory. In this study, the aim was to investigate the effects of simvastatin on concentration, structure and function of molecules of kidney brush border membrane (BBM) by Attenuated Total Reflectance‐Fourier Transform Infrared (ATR‐FTIR) spectroscopy and chemometric analysis.

During 30 days, control and simvastatin treated rats were taken serum physiologic and 50 mg/kg/day simvastatin intragastrically, respectively. Simvastatin‐induced changes in the structure and function of molecules in kidney BBM were determined from the frequency, area and width analysis of spectral bands. Based on these alterations, cluster and principal component analysis (PCA) were applied to discriminate these groups. Moreover, artificial neural network (ANN) analysis was used to determine the secondary structure alterations in proteins

Spectral analysis indicated a decrease in the amount of saturated and unsaturated lipids and proteins with simvastatin treatment. Control and simvastatin groups were successfully distinguished. A significant decrease in beta sheet and increase in random coil content were observed in simvastatin treated group. This study was supported by BAP‐08–11‐DPT‐2011K121010.
A Functional Analysis of the Projection Domain of the Microtubule Associated Protein Tau Using Force Spectroscopy
Authors: Saunders Jared , Donhauser Zachary
Published: June 2021

The microtubule associated proteins (MAP) tau helps stabilize and organize microtubules and plays a role in several neurodegenerative diseases. This study focused specifically on the projection domain of the tau protein, which is a region of the protein that projects outward from the microtubule surface in the tau‐microtubule complex and controls spacing between microtubules in vivo. Our aim was to measure the long‐range repulsive interactions produced by the projection domains of three versions of tau, where this region has been modified, using atomic force microscopy and force spectroscopy. The tau is immobilized on mica surfaces, a model substrate that binds protein in a similar manner to microtubules. We found that as increasingly larger portions of tau's projection domain are removed, the long‐range repulsive interactions decrease. Systematic examination of the behavior of the protein under a range of experimental conditions indicated that a high ionic strength solution essentially removes the long‐range tau‐tau interactions and is consistent with interactions that are primarily electrostatic in nature. Furthermore, we have determined that buffer type does not influence force measurements of the protein, which is evidence that our measurements may be generalized to describe how tau behaves in vivo.
Identification of calregulin binding proteins in a renal cell line
Authors: Alli Abdel A , Ghant Marcus S , Montgomery Darrice S , Eaton Douglas C
Published: June 2021

Calregulin is a calcium buffering protein that regulates calcium storage and release within the endoplasmic reticulum. Calregulin is highly conserved across many species and associates and regulates the function of several proteins. We previously investigated the regulation of MARCKS by calcium/calmodulin. Here, we investigated putative protein binding partners of calregulin at the apical membrane where MARCKS is known to function. Cytoplasmic proteins from HEK293 cells were resolved by two‐dimensional gel electrophoresis. Calregulin was identified among the protein spots by mass spectrometry. Calregulin expression was not limited to the ER but also present in the cytoplasm as demonstrated by sucrose density gradient and Western blot analysis. Calregulin co‐immunoprecipitated with the transmembrane protein, phospholipase C beta‐3, and the actin cytoskeletal protein, spectrin. This interaction required the presence of phosphatase inhibitors, suggesting a requirement for phosphorylation. Future studies will be necessary to identify a potential role for calregulin in regulating the function of phospholipase C beta‐3 and spectrin and calcium binding proteins such as calmodulin.
Mechanism of Non Point Source(S) causing Antibiotic ‐ Resistance in Infectious Diseases
Authors: Kannan Subburaj , Martinez Juan , Montoya Hector , Barrera Jorge , Cooley Roy , Orozco Maria , Villanueva Brenda
Published: June 2021

Self medication, sharing of medication, prescribing antibiotics for viral infection, not completing the dosage, failure to complete the prescribed dosage and excessive use of antibiotics in industrial farming practices, and priming effects are serious public health concerns. Run‐off water from agricultural land and toxic aerosol originating from the recent oil exploration in this region constitute potential Non Point Source for chemical contaminants. The pesticides, suspended sediments, and fertilizers into estuarine habitats adjacent to agricultural areas or downstream from agricultural watersheds may potentially contaminate the aquifer. Subsequent consumption of contaminated water by humans and animal subjects has been has been shown to cause tissue toxicity. Community origin of MRSA (patients with open sores taking bath in common bath tub in the rehabilitation centers), and nosocomial infections are striking realities of this region. Recreational beaches in Southwest Texas, the Gulf coast, and flanking coastal towns in Mexico are a potential non‐point source as reservoir for MRSA (Methicillin‐resistant Staphylococcus aureus). Collectively, aforesaid factors have resulted in a dramatic selection pressure on microbial pathogens carrying antibiotic resistance genes. The inherent nature of AR induced tissue toxicity pose considerable challenges in treating infectious diseases.
Phosphorylation of the eukaryotic cortactin protein by endogenous kinases in an E. coli expression system
Authors: Priester Jacob , Love Haley , Kruchten Anne
Published: June 2021

Purified protein samples are often acquired via recombinant DNA techniques using heterologous E. coli induced expression systems. This bacterial expression and purification technique has been widely utilized for decades, and many researchers have worked under the misconception that eukaryotic proteins are not post‐translationally modified in a significant way during expression and purification in bacteria. Based on contradicting experimental data, we hypothesized that cortactin is phosphorylated by endogenous kinases in E. coli. We analyzed the phosphorylation state of cortactin after in vitro purification from E. coli using western blotting techniques and phosphoprotein stains. We found a heterogenous population of both phosphoserine and phosphotyrosine cortactin after protein purification. As phosphorylation of cortactin has been a significant research focus in many research labs over the last two decades, this result calls for increased scrutiny of experimental results involving in vitro phosphorylated cortactin. This work was supported by the OHSU Medical Research Foundation Award and Linfield College Faculty/Student Collaborative Research Fund.
S1P1 Receptor Regulation by Phosphorylation
Authors: Bazúa‐Valenti Silvana , Morquecho‐León Marco. A , Romero‐Ávila M. Teresa , García‐Sáinz J. Adolfo
Published: June 2021

Sphingosine‐1‐phosphate (S1P) is a bioactive molecule that has been implicated in regulating cellular processes. S1P exerts its extracellular effects as a ligand of a GPCR family. S1P1 receptors are coupled to heterotrimeric Gi/o proteins. The importance of S1P and its receptor S1P1 in multiple cellular and physiological processes (vascular tone, permeability, angiogenesis, lymphocyte egress) makes them and their regulation, an interesting pharmacological target. It has been demonstrated that S1P1 activation by its natural ligand and the activation of PKC by phorbol myristate acetate leads to receptor phosphorylation and internalization. However, little is known about the identity of the kinases involved in this process. The primary objective of this study is to characterize the molecular processes that lead to receptor phosphorylation and identify the PKC isoform/s implicated.

A human prostate cancer cell line stably expressing the S1P1 fused to the Green Fluorescence Protein was used for this work. We conducted functional analysis by measuring intracellular calcium mobilization in the presence or absence of PMA and distinct PKC selective inhibitors for the different isoforms. S1P1‐PKC interaction in the course of stimulation with PMA was analyzed by co‐immunoprecipitation. Our results indicate that of two isoforms participate in the PMA‐mediated desensitization, PKC α and to a lesser extent PKC β.
Characterization of a microtubule‐associated protein, doublecortin (DCX), as a substrate of c‐Jun N‐terminal Kinases (JNKs)
Authors: Ngoei Kevin , Gooley Paul R , Parker Michael W , Bogoyevitch Marie A
Published: June 2021

We are interested how docking motifs contribute to substrate recognition by JNK, a Ser/Thr protein kinase and member of the Mitogen‐Activated Protein Kinase (MAPK) family. The activation of JNK signaling pathways and concomitant phosphorylation of a range of JNK substrates has been implicated with regulation of critical events, including cell growth and proliferation, cell division and cell death. Specifically, we are defining the structural and functional properties of the microtubule‐associated protein doublecortin (DCX) as a JNK substrate. DCX is unusual with its evolutionary conserved DC domains, rather than a typical linear basic/hydrophobic motif, reported to mediate its interaction with JNK. Using mass spectrometry, we have identified a novel JNK‐mediated phosphorylation site in the DCX protein, and confirmed the phosphorylation of this site by mutagenesis and detection by phosphospecific antibodies. Through our use of NMR approaches, we are gaining new structural insights into DCX with the ultimate aim to identify the features of the JNK‐DC domain interaction interface that support this non‐conventional mode of JNK‐substrate recognition.