bluuebunny/crossref_metadata_2025_split huggingface dataset:
ARRDC5 cooperates with ACAP4 to regulate ARF6 activity at the cell membrane (964.1)
Authors: Zhou Jiajia , Liu Hongsheng , Zhao Xuannv , Wang Dongmei , Wang Fengsong , Liu Fusheng , Yao Xuebiao 
Published: June 2021

The ARF6 GTPase is essential for regulating multiple cellular processes, such as, secretion, endocytosis, membrane trafficking, cell migration. ACAP4 is an ARF6 GTPase‐activating protein is critical EGF‐elicited cell migration (Mol. Cell Proteomics. 5, 1437‐49). However, how ACAP4 regulates proper levers of active ARF6 is not well understood. Here we show that, arrestin domain‐containing 5 (ARRDC5), a novel protein of α‐ arrestin family, competes with ARF6 for binding ACAP4. ARRDC5 can inhibit ACAP4 mediated inactivation of ARF6 in a dose‐dependent manner. Knock‐down of ARRDC5 decreases cellular ARF6‐GTP, whereas ARRDC5 overexpression increases cellular ARF6‐GTP. Surprisingly, EGF stimulation of cells causes phosphorylation of ARRDC5 at Ser247 by AKT1, which is essential for cell motility. Significantly, our biochemical characterization demonstrated that the phosphorylation of Ser247 strengthens ACAP4‐ARRDC5 association. The co‐localization of ACAP4 with ARRDC5 occurred in ruffling membranes formed upon EGF stimulation. In addition, expression of the phospho‐mimicking mutant of ARRDC5 promotes ARF6 activation and cell migration, knock‐down of AKT1 inhibits ARF6 activation and cell migration. Our date thus uncover ARRDC5 as a modifier of cellular ARF6‐GTP through regulation of ACAP4. AKT1‐mediated ARRDC5 phosphorylation facilitates cell migration by sequestering ACAP4 at the cell membrane to stabilize active ARF6 in response to EGF stimulation.Grant Funding Source: DK56292
Progress toward a novel pro‐drug approach for temporal manipulation of protein kinase A with a commercially available ruthenium catalyst (968.1)
Authors: Hunt Bethany , Dahlen Shelby , Streu Craig , Mertz Pamela 
Published: June 2021

Transition metal catalysts have been demonstrated in principle for temporal and spatial control of reactions, but this technology has yet to be broadly applied as a tool for biological study and cellular manipulation. Here we demonstrate the use of a ruthenium complex catalyst as a novel approach for spatial and temporal release of a pro‐drug inside cells, namely the transition of caged H89 to active H89, a Protein Kinase A (PKA) inhibitor. We synthesized caged H89 by protecting H89 as an allyl carbamate. We designed the system to intracellularly generate an active H89 inhibitor from caged H89 following addition of the ruthenium metal catalyst with the goal of inhibiting the catalytic activity of PKA and therefore reducing the phosphorylation of perilipin 1. The ruthenium catalyst activity was monitored by assessing the phosphorylation of perilipin 1, which is phosphorylated by PKA on 6 serine residues and not phosphorylated by other kinases. Experiments were performed on cell lines expressing endogenous perlipin 1, differentiated 3T3L1 cells and Y1 adrenal cells. Our results show that in its active state, H89 inhibited phosphorylation activity of PKA on substrates such as perilipin whereas the caged form of H89 lacked inhibitory activity. Experiments are planned for use of the ruthenium catalyst after the H89 and caged H89 incubation steps to determine the ability of the catalyst to activate caged H89 intracellularly.Grant Funding Source: Supported by The Royal Society of Chemistry
Dynamic elucidation of drug transport pathways in human P‐glycoprotein (974.5)
Authors: McCormick James , Vogel Pia , Wise John 
Published: June 2021

Chemotherapy resistant, recurrent cancers most commonly express members of the ABCB1 class of plasma membrane transporters, specifically MDR1 P‐glycoprotein transporter (P‐gp). At high levels of expression, the P‐gp transporter lowers the intracellular concentrations of chemotherapeutics to below effective therapeutic levels. Because P‐gp can transport a broad range of substrates, such expression leads to general multidrug resistance in the population of cancerous cells. Dynamic P‐gp models which transition through putative catalytic cycles have been previously created by us using targeted molecular dynamics simulations (TMD) with four different crystallographic conformations of related proteins. In the present study, these simulations have been expanded to include transport substrates docked into different initial sites within the drug binding domain of P‐gp. Even though drug movement in these simulations was not targeted by TMD, transport of chemotherapeutics (daunorubicin and doxorubicin) and a P‐gp transport substrate (verapamil) through the membrane by human P‐gp was observed during the putative catalytic cycles. Initial results suggest that different starting positions of transport substrate in the “open to the cytoplasm” conformation resolve into a common transport pathway through the membrane as P‐gp closes to the cytoplasm and opens to the extracellular space.Grant Funding Source: NIH 1R15GM094771 ‐ 01A1, Communities Foundations of TX
The mycobacterial pupylation pathway (98.2)
Authors: Weber‐Ban Eilika , Barandun Jonas , Delley Cyrille 
Published: June 2021

Mycobacteria are prominent members of the actinobacterial phylum owing to the notoriety of the pathogen Mycobacterium tuberculosis (Mtb). The mycobacterial habitat is subject to rapid changes in nutritional sources, requiring from the bacteria a multitude of adaptive mechanisms including protein degradation. Three energy‐dependent protease systems exist in Mtb, the membrane‐associated FtsH, the ClpP protease and a bacterial proteasome assembly restricted to actinobacteria. Substrates are targeted to the proteasome by a post‐translational modification akin to ubiquitination, termed pupylation (1). Pupylation involves the covalent attachment of Pup (prokaryotic ubiquitin‐like protein) to lysine residues of the target and requires the sequential action of two enzymes, the deamidase Dop (deamidase of Pup) and the Pup ligase PafA (2). First, Dop renders Pup ligation‐competent by conversion of its C‐terminal glutamine to glutamate. Then PafA catalyzes the covalent attachment of Pup’s C‐terminal glutamate to the target lysine. We study the Pup‐proteasome pathway by biochemical and structural techniques with particular focus on how the intrinsically disordered Pup is recognized by the pupylation enzymes and how target proteins are selected for modification.In Mtb, the Pup‐proteasome system contributes to pathogenicity by supporting the persistence within macrophages. The components of pupylation are thus attractive targets for drug development. Understanding their recognition determinants and mechanism of action provides the necessary molecular framework.(1) Pearce (2008) Science 322: 1104.(2) Striebel (2009) NSMB 16: 647.
Comprehensive cancer cell proteomics: a global counterpoint to genomic techniques (981.7)
Authors: Bakalarski Corey , Lianoglou Steve , Neve Richard , Yu Mamie , Selvaraj Suresh , Possematto Anthony , Beausoleil Sean , Yue Peng , Dornan David , Bourgon Richard , Forrest William , Kirkpatrick Donald 
Published: June 2021

Next‐generation genomic data has produced profound insights into the genetic mechanisms of human disease. However, little is known about how rigidly these genetic features translate to protein abundance and activity. Here, we leveraged tandem mass tagging (TMT 6‐plex) proteomics to build a quantitative map of baseline protein abundance across hundreds of cell lines important to human disease.Whole cell lysates from 189 cell lines were digested, chemically labeled and analyzed with an LTQ‐Orbitrap Velos mass spectrometer using an HCD‐MS3 method. In all, >13 million quantitative measurements were collected from 2.2 million peptide‐spectral matches. Quantitative data were normalized using a linear mixed effects model to yield relative protein abundances for >8,500 proteins and >100,000 unique peptides.Comparisons to RNASeq revealed extensive correlation between transcript expression and protein abundance both overall and in genes with known amplification events (ERBB2, EGFR, MET). Poorly correlated expression/abundance levels for a subset of proteins revealed instances of alternate events such as mutational state (TP53) or copy number variation driving protein abundance. Ongoing work seeks to correlate these results with sensitivity/resistance to therapeutic agents.
The human kinase‐substrate phosphorylation network rewired by genetic polymorphisms is heavily associated with cancers (984.3)
Authors: Xue Yu , Wang Yongbo , Liu Zexian 
Published: June 2021

Large‐scale sequencing has characterized an enormous number of genetic variations (GVs), and the functional analysis of GVs is fundamental for understanding the difference of disease susceptibility and therapeutic response among populations. Combined with a sequence‐based predictor of GPS 2.1 (http://gps.biocuckoo.org), known phosphorylation and protein‐protein interaction information, we computationally detected 4,490 potential phosSNPs (Phosphorylation‐related non‐synonymous single nucleotide polymorphisms) including 275 known disease‐associated SNPs that dramatically rewire the human phosSNP‐associated kinase‐substrate network. Also, cancer genes and drug targets were significantly over‐represented in the network, whereas proteins in the network are heavily implicated in a number of signaling and cancer pathways. Moreover, we observed that known inherited disease‐ or cancer‐associated mutations were significantly over‐represented in predicted phosSNPs, with a two‐fold enrichment. In addition, we re‐constructed four population‐specific kinase‐substrate networks and revealed several inherited disease or cancer genes such as IRS1, RAF1 and EGFR were differentially regulated by phosSNPs. Thus, phosSNPs may influence the disease and cancer susceptibility by differentially rewiring the phosphorylation networks among different populations. Taken together, our results can be helpful for further experimental analysis of the molecular mechanisms of known disease‐associated phosSNPs, and provided new leads for personalized medicine. The PhosSNP 2.0 database was freely available for academic research at http://phossnp.biocuckoo.org.Grant Funding Source: Supported by Natural Science Foundation of China (31171263, 81272578)
Genesis of antibiotic resistance (AR) III: trifling risk of AR pathogens induced infectious diseases from regulated concentrated animal feeding operations (986.3)
Authors: Kannan Subburaj , Hernandez Lizeth , Herrera Ana , Jimenez Brenda , Miller Monica , Perales Patricia , Reyna Cristal , Subburaj Prabavathy 
Published: June 2021

Concentrated Animal Feeding Operations (CAFOs) are sites where animals are housed and raised in a confined environment. CAFOs assemble animals that are fed and cared for by professional care givers referred to as “Cowboys” on a limited space in a rustic landscape. The operation is monitored and meets the standards set forth by The United states Environmental Protection Agency (http://www.epa.gov/region7/water/cafo/). In southwestern Texas, cattle are imported from Mexico, and subjected to the health inspection by The United States ‐ Mexico border USDA‐APHIS‐VS stations. Cattle undergo rigorous inspections for Tuberculosis, tick‐borne infections, and respiratory illness, along with prior health records followed by consignment to CAFOs (Feed Lots). While in Feed lots, the health and well being of the cattle is monitored by the house staff and also by USDA veterinarian. The amount of “Antibiotics” supplemented in the cattle feed is regulated by the EPA standards and followed through by in house nutritionist. MICRO BEEF TECHNOLOGIES, Ltd (http://www.microbeef.com/about_micro/index.html ) provides an excellent tracking system to maintain comprehensive details on individual animals regarding their health and nutrition. Taken together with an exceptional monitoring programs by The US‐EPA, USDA‐APHIS‐VS, and strict industry standards upheld by The Texas Cattle Feeders Association, the CAFOs pose minimal risk of rendering AR pathogen affected infectious diseases. (Acknowledgement: Dr. Darrell Haney, USDA‐APHIS‐VS, (830) 752‐1785, Eagle Pass, Texas; Mr. Luis De La Cruz, Runnells‐Peters Feed Yard LLC, 1444 Fm 1665, Quemado, TX 78877‐7790; Information Resources from Texas Cattle Feeders Association, http://www.tcfa.org/SiteMap.html).Grant Funding Source: Professional Development Funds ‐ SWTJC, Eagle Pass, Texas 78852
Evidence for an interdomain flavin binding site in Steap (six‐transmembrane epithelial antigen of the prostate) family metalloreductases associated with iron homeostasis and insulin resistance (996.4)
Authors: Kleven Mark , Gauss George , Fleming Mark , Lawrence C. 
Published: June 2021

Steap family enzymes are integral membrane NADPH oxidases capable of reducing both Fe3+ and Cu2+ at the cell surface. While Steap3 has previously been identified as the ferrireductase in the transferrin cycle and Steap4 is linked to obesity associated insulin resistance, cellular roles and chemical underpinnings of the family remain ill‐defined. Here we report in‐depth biochemical and structural characterization of Steap3 and Steap4. Consistent with both cell surface and endosomal activities, they show significant metalloreductase activity over a broad pH range, and recognize both Fe3+ and Cu2+ at physiologically relevant concentrations. Structure‐function studies also reveal the absence of a high affinity flavin binding site in the isolated N‐terminal oxidoreductase domains. Full‐length proteins, however, show high affinity binding sites for FAD. This suggests that Steap family members may also function in copper transport, and that an interdomain flavin binding facilitates electron transfer between NADPH of the oxidoreductase domain, and a heme cofactor in the transmembrane domain. In addition, bimolecular fluorescence complementation (BiFC) indicates they are active as homodimers.Grant Funding Source: Supported by the National Institutes of Health, Grant #: GM084326
Cellular zinc levels are influenced by the interaction between the mucolipidosis IV‐associated TRPML1 protein and TMEM163 protein (996.5)
Authors: Silva Joshua , Basilio Luigi , Hart Thomas , Tringali Jonathan , Cuajungco Math 
Published: June 2021

Dysfunctional Mucolipin‐1 (TRPML1) ion channel protein causes the human lysosomal storage disorder Mucolipidosis type IV (MLIV). Cells affected by MLIV have significantly higher intracellular zinc levels compared to normal cells; however, the mechanism of zinc elevation remains unknown. Using a membrane‐based yeast two‐hybrid technique, we identified a putative zinc‐binding protein called transmembrane‐163 protein (TMEM163) as an interacting partner for TRPML1. We confirmed interaction by co‐immunoprecipitation and mass spectrometry. We hypothesized that the interaction between TMEM163 and TRPML1 plays a role in regulating TMEM163, and thus intracellular zinc. RNA interference targeting both TMEM163 and TRPML1 in SH‐SY5Y neuroblastoma cells resulted in significant increase of intracellular zinc when both proteins were knocked down. To determine how interaction of TRPML1 and TMEM163 could be responsible in regulation of intracellular zinc, we co‐expressed and analyzed expression of tagged constructs of both proteins using human embryonic kidney (HEK)‐293 cells. Evidence from cell surface biotinylation showed that co‐expressed TRPML1 resulted in reduced TMEM163 localized within the plasma membrane of HEK‐293 cells. This result possibly explains the reduction of cellular zinc flux when TRPML1 is co‐expressed with TMEM163. Overall, our data suggest that both TRPML1 and TMEM163 proteins play a role in intracellular zinc regulation. Thus, the loss of TRPML1 protein function and/or the reduction of TMEM163 protein could result in zinc dyshomeostasis in MLIV.Grant Funding Source: Supported by NIH R15‐NS070774 and CSUF Intramural Grants
Cross‐linking study of membrane subunits K, L, M and N in E. coli complex I (997.10)
Authors: Vik Steven , Zhu Shaotong , Tursun Ablat 
Published: June 2021

Complex I is an entry point of the electron transport chain and plays a major role in proton gradient generation. Oxidation of NADH, reduction of ubiquinone, and proton translocation are three essential functions of Complex I. Subunits L, M, and N, the three largest subunits in the membrane domain, are homologous to components of multi‐subunit Na+/H+ antiporters, so they are implicated in proton pumping. Based on available evidence it is believed that a redox‐linked conformational change is necessary for proton pumping. According to recent crystal structures, subunit L contains an unusual helix HL extending laterally along the membrane surface from subunit L, past subunit M, and to subunits N and K. In this study, cross‐linking of engineered cysteine residues using disulfide formation and bi‐functional reagents was carried out to detect regions of the proton transporter that have restricted motion. Fourteen cysteine pairs between helix HL and membrane subunits L, M, N and K were cross‐linked and none showed no significant loss of enzyme activity or proton pumping These results suggest that there is not significant movement of the lateral helix. Further cross‐linking studies were done between pairs of cysteine residues within subunit N. The results of those studies suggested possible proton pathways.Grant Funding Source: NIH 1R15GM099014
Identification of trafficking motifs in the C‐terminus of the cystine/gluatamate exchanger, System xc‐ (997.3)
Authors: Ladd Nicole , Unterbrink Elizabeth , Georges Anne , Lang Sara , Chase Leah 
Published: June 2021

System xc‐ is a heterodimeric plasma membrane transporter, comprised of the proteins xCT and CD98, which facilitates the stoichiometric exchange of extracellular cystine for intracellular glutamate. System xc‐ serves to protect the cell from oxidative stress, and brief exposure of cells to oxidative agents leads to an increase in trafficking of xCT to the plasma membrane. However, little is known about the mechanisms which govern the constitutive and regulated trafficking of xCT. In the present study, we sought to identify trafficking motifs within the C‐terminus of xCT that are important in regulating the delivery and internalization of xCT from the membrane. We used a mutagenesis approach combined with cell surface biotinylation assays to identify regions of the C‐terminus that are involved in the constitutive trafficking of xCT. Specifically, using site‐directed mutagenesis, we identified a tyrosine based motif between positions 464 ‐486 containing the sequence YYFII that contributes to the constitutive trafficking of xCT to the plasma membrane. In addition, we created three truncation mutants at positions 469, 484, and 496 of xCT, and found that an endocytic motif(s) must also exist between position 469 and 484. While there are no canonical endocytic motifs within this region, there is a putative ubiquitination site. Since we have recently demonstrated that xCT is ubiquitinated in Cos‐7 cells and ubiquitination of membrane proteins is known to regulate cell surface expression, we are currently using site‐directed mutatgenesis to test the hypothesis that ubiquitination of xCT in this region leads to the increased rate of internalization of xCT. This research was supported in part by NSF‐RUI #0848564.Grant Funding Source: NSF‐RUI #0848564
Single molecule studies of the E.coli protein export system (997.9)
Authors: Sanganna Gari Raghavendar Reddy , Frey Nathan , Mao Chunfeng , Randall Linda , King Gavin 
Published: June 2021

The general secretory or Sec system is the major route of export for proteins from the cytosol of Escherichia coli. The pathway through the membrane is provided by the translocon SecYEG. At the membrane, ATPase SecA binds SecYEG and drives protein translocation. In this work we shed light on Sec system using atomic force microscopy (AFM), a single molecule technique that is well suited for studying membrane proteins in near native conditions. AFM images provided direct visualizations of the dynamic loops of SecYEG which orchestrate critical translocon functions as well as determination of the oligomeric state of SecYEG in lipid bilayers. Additionally, we studied structure‐function relationships of SecA‐SecYEG complexes. Our results show that SecA binds SecYEG in liposomes in two distinct modes. When SecA was added extraneously to liposomes containing SecYEG, the proteins showed a wide distribution of heights with no prominent peaks, indicating that SecA populates multiple binding states. In contrast to this broad distribution of heights, the sample of proteoliposomes in which SecY and SecA were co‐assembled had a predominant species centered at 40 Å in height above the lipid bilayer. Because these proteoliposomes contain approximately sixfold more active translocons than do the proteoliposomes to which SecA was added after assembly, we conclude that this structural state represents SecA bound to translocons that have been rendered active. Taken together this work provides new functional and structural insights into the Sec system. More generally, the assays developed here are adaptable to other membrane protein systems.Grant Funding Source: NSF CAREER Award , Burroughs Wellcome Fund Career Award, NIH
Phosphatidylinositol‐4‐phosphate synthesis and turnover spans multiple membrane compartments (999.2)
Authors: Hammond Gerry , Machner Matthias , Balla Tamas 
Published: June 2021

Organelle identity may be assisted by a phosphoinositide (PI) code, wherein specific PI lipids act as molecular “signposts”, ensuring exclusive recruitment of binding proteins to the correct membrane compartment. One example is PtdIns4P, whose known effector proteins are recruited to the Golgi. However, the PI 4‐kinases (PI4K) that make PtdIns4P belie such a restricted function, since they act at the Golgi, plasma membrane (PM) and in lysosomal traffic. To reconcile this contradiction, we re‐examined the distribution of PtdIns4P with a novel biosensor derived from the P4M domain of L. pneumophila SidM. P4M localizes to the Golgi, PM and dynamic Rab7‐positive late endosomes/lysosomes. Chemically‐induced recruitment of PI phosphatases to these compartments demonstrates PtdIns4P is the lipid bound in each case. Whereas the bulk of another PI lipid, PtdIns(4,5)P2 is localized at the PM, it could be produced in the other PtdIns4P‐containing compartments by recruitment of a PtdIns4P 5‐kinase. P4M can also detect ectopically produced PtdIns4P on ER‐derived membranes. We show that each of the four PI4Ks overlap with pools of PtdIns4P, and the ER/Golgi localized PtdIns4P phosphatase, Sacm1l, hydrolyses both Golgi and PM pools. We conclude that PtdIns4P has a much more complex function than as a simple molecular signpost for the Golgi, and likely functions through several as yet unappreciated protein interactions.Grant Funding Source: Intramural Research Program of the Eunice Kennedy Shriver NICHD, NIH