bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of CRX Protein Profile in the Rdy Cat Model Suggests Molecular Mechanism of Pathology
Authors: Deckman Koren Holland , Dempster Katrina , Khalsa Amrit , David Victor , O'Brien Stephen J , Narfström Kristina , Menotti‐Raymond Marilyn
Published: June 2021

The product of the cone‐rod homeobox gene, CRX, is an important transcription factor critical in the development and maintenance of photoreceptor cells in the retina. Mutations have been document in the human CRX gene that can lead to retinal pathologies such as cone‐rod dystrophy (CoRD). A CRX gene mutation was recently characterized the Rdy (rod‐cone dysplasia) cat, which exhibits an early onset, autosomal dominant disorder modeling human CoRD. A single base deletion in exon 4 resulted in the introduction of a premature stop codon and one‐third of the CRX protein, including the transcriptional transactivation domains, is deleted. A truncated CRX product, as well as the full‐length CRX product, are translated and persist in Rdy individuals as detected through Western blot analysis. Preliminary electrophoretic mobility shift assays suggest that the truncated product binds to the promoter site of feline rhodopsin, likely acting as a competitive inhibitor of the wildtype product. The Rdy cat will provide valuable insights into the mechanisms causative of Rdy disease pathology and is an important animal model of human retinal disease.
Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using Targeted Approach with a Hybrid Linear Ion Trap Orbitrap Mass Spectrometer
Authors: Kiyonami Reiko , Prakash Amol , Zabrouskov Vlad
Published: June 2021

High‐resolution, accurate‐mass (HR/AM) mass spectrometry is routinely used for identification and relative quantitation of peptides present in complex mixtures using stable‐isotope labeling, isobaric tags, or label‐free techniques. However, HR/AM application for targeted peptide analysis is less explored. Due to the complexity of biological samples, accurate mass and retention time alone are not sufficient for verifying identity of a targeted peptide. Therefore, sequence confirmation via MS/MS is essential during the early verification and validation stages of development of a targeted quantitative assay. Here, a workflow was developed for targeted protein/peptide quantitation on the LTQ Orbitrap Velos to use high resolution selected ion monitoring (SIM) for quantitation followed by targeted MS/MS for sequence confirmation. A standard peptide mixture spiked into a yeast digest mixture at different concentration as quantitative targets and LOD was as low as 10 amol. An additional yeast digest was used to further evaluate the precision and throughput and the workflow by 77 peptides (25 yeast proteins). All the peptides were successfully quantified and verified with high analytical precision in a single LC/MS run.
Microbial Diversity in Soil Assessed by Two Different Capillary Electrophoresis Methods
Authors: Patel Niraj S , Larson Sabreena A , Carter David O , Drijber Rhae A , Bailey Cheryl P
Published: June 2021

Capillary electrophoresis can separate fragments of nucleic acids using single strand conformation polymorphism (SSCP) and terminal restriction fragment length polymorphism (T‐RFLP). These methods have been increasingly used in microbial ecology to develop fingerprints of the microbial community within soil. T‐RFLP is based on amplifying a region of DNA common to multiple species using conserved PCR primers whereas SSCP detects mutations in DNA fragments due to changes in the secondary structure of single‐stranded DNA fragments. The objective of this study is to use soil samples to compare molecular profiles generated by CE‐SSCP and T‐RFLP in detail. The soil samples will be collected from four locations representing four soil types. One set of soil subsamples will be analyzed immediately following collection and six subsamples will be placed in different storage treatments: cooling at −4C, freezing at −20C, freezing at −80C, freeze drying, air drying, and oven drying. DNA will be extracted from the samples, run through polymerase chain reaction (PCR) to amplify and fluorescently tag the genes of interest. T‐RFLP uses restriction enzyme MspI after PCR, while SSCP does not. Then, the microbial community fingerprints are analyzed. We hypothesize CE‐SSCP and T‐RFLP will generate similar fingerprints of the microbial community within soil. Supported by National Institute of Justice (25‐6228‐0159‐001).
Molecular mechanisms involved in the antithrombotic activity of aegyptin, a novel mosquito‐derived collagen‐binding protein
Authors: de Moraes Mizurini Daniella , Calvo Eric , Francischetti Ivo , Monteiro Robson
Published: June 2021

Aegyptin is a 30‐kDa mosquito salivary gland protein that binds to collagen with high affinity (Kd=6 nM) and inhibits platelet aggregation. It recognizes the von Willebrand factor binding‐site in collagen which could partially explain its biological activity. Herein we evaluated: 1. whether this protein could prevent in vitro collagen‐induced plasma clotting; 2. the in vivo antithrombotic activity of aegyptin. Aegyptin does not affect collagen‐mediated acceleration of plasma clotting. In accordance with this observation, aegyptin has no effect on collagen‐induced FXII activation, as indirectly determined by measurement of kallikrein formation in plasma. Aegyptin has no antithrombotic activity in vivo by employing an arteriovenous shunt model in rats. This model is independent of collagen‐exposure since presents no vascular surface for thrombus formation. On the other hand, aegyptin delays laser‐induced carotid thrombus formation in the presence of Rose Bengal, a model based on vessel injury. Our results demonstrate that aegyptin is an effective antithrombotic agent in vivo and might be regard as an important tool to dissect the hemostatic role of collagen in vivo.
Molecular bases of disease expression of syndromic visual dystrophies by RPGR‐dependent and differential subcellular sorting and processing of RPGRIP1
Authors: Ferreira Paulo A , Cho Kyoungin , Yi Haiqing , Guruju Mallikarjuna
Published: June 2021

RPGR and RPGRIP1 play direct roles in syndromic retinal dystrophies by elusive mechanisms. RPGR interacts with the RPGRIP1 via its RHD domain, which is homologous to RCC1, a nucleotide‐exchange factor for RAN GTPase. Molecular modeling of RHD of RPGR to RCC1 shows that all disease mutations in RHD map to a distinct contact interface from that found between RCC1 and RAN GTPase. Two‐hybrid assays show that disease‐causing mutations in RHD or ORF15 domains differentially impair RPGR interaction with RPGRIP1. Cell and time‐lapse microscopy assays support that expression of RPGRIP1α1 isoform alone promotes the genesis of aggregates, whereas its co‐expression with the RPGR1–19 isoform targets RPGRIP1α1 to the Golgi. Conversely, RPGRORF15 co‐expression with RPGRIP1α1 promotes its pan intracellular dispersion and clears out pre‐existing RPGRIP1α1 deposits. Mutations singly in Rpgr1–19, RpgrORF15 or Rpgrip1, do not affect the colocalization of RPGR1–19 or RPGRORF15 with RPGRIP1α1, whereas co‐expression of similar mutations in Rpgr variants and Rpgrip1 abrogates their colocalization and association. RPGRORF15, but not RPGR1–19, prevents the C‐terminal proteolytic processing of RPGRIP1α1. Hence, Rpgr1–19 and RpgrORF15 exert distinct effects on RPGRIP1α1 sorting and processing and they are necessary but not sufficient to the coupling to, and subcellular targeting of, RPGRIP1α1.

NIH support GM083165 & EY019492
Smooth muscle cell expression of a constitutive active form of human Profilin1 accelerates cutaneous wound repair
Authors: Abouelnaga Zeinb A , Hassona Mohamed DH , Elnakish Mohammad T , Alhaj Mazin , Hassanain Hamdy
Published: June 2021

The repair of wounds is one of the most complex biological processes. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. Wounding generates a zone of actin polymerization around the wound. Actin polymerization plays an important role in wound closure; In the process of healing, F‐actin fingers on opposing sides of the wound contact each other and pull the wound edges toward each other. One of the most important regulators of F‐actin dynamics is profilin1. We determined the role of profilin1protein in the wound healing process by using profilin1 and 88R/L transgenic mice. At post wound day 3, 6 and 9 (PWD 3, 6, 9), we found a significant decrease in the wound size of profilin1 mice (52, 25, 17%, respectively) compared to nontransgenic and 88R/L mice. Additionally, there was an increase in vascularization and cell migration in the profilin1 mice wound area at PWD 6 compared to nontransgenic and 88R/L mice. Western blot analyses showed a significant increase in collagen III (412%), α1 (374%) β1(311%) integrins, SRF (380%), Rac1 (289%) and HIF1α (323%) in the profilin1 mice wound area at PWD 7 compared to nontransgenic mice. Moreover, there was a significant increase in hair follicle formation, collagen IV and VEGF expression in profilin1 mice wound area compared to control at PWD 7. In conclusion, profilin1 is an unexpected molecule responsible for the closure of the wound.
Effects of BIG1 and KANK1 on cell polarity and directed migration during wound healing
Authors: LI CHUN‐CHUN , Kuo Jean‐Cheng , Kiyama Ryoiti , Moss Joel , Vaughan Martha
Published: June 2021

Brefeldin A‐inhibited guanine nucleotide‐exchange protein (BIG)1 activates class I ADP‐ribosylation factors by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus‐end‐directed motor protein, moves cargo away from the microtubule‐organizing center (MTOC) on microtubules. Here, we describe a newly recognized relationship between BIG1 and KANK1, a protein that has multiple actions in cells and interacts with KIF21A. Microscopically, overexpressed KANK1 appeared partially colocalized with endogenous BIG1, consistent with potentially functional interactions as was their reciprocal immunoprecipitation (IP), but direct interaction of KANK1 and BIG1 was not established. Depletion or overexpression BIG1 protein appeared not to affect KANK1 distribution. Effects of BIG1‐ and KANK1‐depletion on cell migration in wound‐healing assays were, however, remarkably similar. Depletion of BIG1 or KANK1 interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, but KIF21A depletion did not have such effects. These studies identify novel functions of both BIG1 and KANK1 in regulating cell polarity during directed migration.
Disruption of PKB Signaling Restores Chemotaxis of Cells Lacking Tumor Suppressor PTEN
Authors: tang michelle , Iijima Miho , Kamimura Yoichiro , Chen Lingfeng , Long Yu , Devreotes Peter
Published: June 2021

By limiting phosphotidylinositol 3,4,5‐triphosphate(PIP3) levels, tumor suppressor PTEN not only controls cell growth but also maintains cell polarity required for cytokinesis and chemotaxis. To identify the critical targets of PIP3 that link it to the cytoskeleton, we deleted secondary genes to reverse the deficiencies of pten− cells in Dictyostelium. The polarity defects in pten− cells correlate with elevated phosphorylations of PKB substrates. Deletion of AKT orthologue, PkbA, or a subunit of its activator TORC2, reduced the phosphorylations and suppressed the cytokinesis and chemotaxis defects in pten− cells. In these double mutants, the excessive PIP3 levels and, presumably, activation of other PIP3‐binding proteins had little or no effect on the cytoskeleton. In bands with increased phosphorylation in pten− cells, we found PKB substrates, PI5K, GefS, GacG, and PakA. Disruption of PakA in pten− cells restored a large fraction of the cells to normal behavior. Consistently, expression of phosphomimetic PakA in pten− cells exacerbated the defects but non‐phosphorylatable PakA had no effect. Thus, among many putative PTEN‐ and PIP3‐dependent events, phosphorylation of PKB substrates is the key downstream regulator of cell polarity.
cAMP signal relay and streaming during Dictyostelium development
Authors: Das Satarupa , Bagorda Anna , Rericha Erin , Parent Carole
Published: June 2021

Upon starvation, Dictyostelium discoideum cells enter a developmental program leading to a transition between cells operating as individuals to group behavior. The process is mediated by extracellular cyclic AMP (cAMP) binding to the cAMP receptor 1 (cAR1), which initiates a signaling cascade leading to the activation of Adenylyl Cyclase A (ACA). A burst of cAMP is synthesized when ACA is first activated. While part of the cAMP synthesized remains inside cells to activate PKA, the majority of it is secreted to act in an autocrine and paracrine fashion. For a spatially extended cell population, release of cAMP allows neighboring cells to polarize and migrate directionally, a process known as chemotaxis, and form characteristic chains of cells called streams. We now report that cAMP relay can be readily measured biochemically by assessing ACA activity at various time points after stimulating cells with subsaturating concentrations of cAMP. We also find that the relay of cAMP signals can be measured by monitoring ERK2 or PKBR1 activation/deactivation. Using cells with high intracellular cAMP, we further establish that the capability of cells to relay signals changes during development – a phenomenon that occurs coincidently with the streaming ability of cells during chemotaxis. We propose that as cells proceed through development, cAMP sequesters cAR1, which dramatically impacts downstream signaling cascades.
G‐protein signaling and adaptation in chemotaxis
Authors: Huang Chuan‐Hsiang , Devreotes Peter N
Published: June 2021

Chemotaxis, the directed migration of cells in chemical gradients, is a fundamental phenomenon in many types of cells. In Dictyostelium, the chemoattractant cAMP triggers G‐protein coupled receptor signaling mediated by the heterotrimeric Gα2/Gβ/Gγ complex. Using FRET between Gα2‐CFP and Gβ‐YFP as readout, activation of G protein can be monitored in real time. cAMP stimulation leads to a rapid decrease in FRET due to the dissociation of Gα2‐CFP and Gβ‐YFP, whereas removal of stimulus causes a slow recovery of FRET. The different kinetics of FRET loss and recovery is readily explained by the G‐protein cycle model. However, if FRET loss reflects G‐protein activation, the slow recovery would cause delocalized signals, which compromise gradient sensing during chemotaxis. Furthermore, expression of a constitutively active mutant of G‐protein in Dictyostelium leads to persistent activation of downstream proteins. These results suggest that G‐protein may be inactivated in the dissociated state.
Micropatterned substrates: Tools for studying cell motility and aiding rational drug design
Authors: Huda Sabil , Soh Siowling , Pilans Didzis , Mahmud Goher , Kandere‐Grzybowska Kristiana , Grzybowski Bartosz A.
Published: June 2021

We have developed a novel class of cell biology tools for the quantification of cytoskeletal organization and dynamics in populations of cancerous cells with low variability. Anisotropic Solid Microetching (ASoMiC) was used to constrain cells onto transparent microislands and subsequently imaged. This methodology creates a unique platform for studying cell motility and identifying therapeutic targets.

Triangular microislands spatially segregate cytoskeletal components and allow for study of microtubule (MT) growth trajectories in defined geometric confines. We show that growing MTs are targeted towards focal adhesions (FAs). RNAi studies reveal that myosin IIA dependent f‐actin bundles are integral to this process. Since MT targeting of FAs is important in metastasizing cancer cells, this system can be used to identify new cancer therapeutic targets.

To study cell motility, we used micropatterned linear tracks to constrain cells to 1‐D. We found that metastatic cells were “superdiffusive” vs. “diffusive” non‐metastatic cells. This quantitative description of a cell's motility leads to the systematic characterization of metastatic potential and response to therapeutic agents.

ASoMiC allows the study of cytoskeletal organization and dynamics of cancerous cells with minimal cell‐to‐cell variability. These systems provide a platform for the study and manipulation of the cytoskeletal organization.

Funding: NIH Award 1R21CA137707‐01
PDGF‐BB induces the cell surface co‐localization of laminin and dystroglycan and fibronectin and α5‐integrin
Authors: Kastanis George John , Hernandez‐Nazara Zamira , Nieto Natalia , Rincón‐Gallardo Ana Rosa , Popratiloff Anastas , Dominguez‐Rosales Jose Alfredo , Lechuga Carmen , Rojkind Marcos
Published: June 2021

Hepatic stellate cells (HSC) are embedded in a laminin‐rich matrix in the Space of Disse of the normal liver. During injury HSC migrate to the injured sites and produce scar collagen. One of the first proteins produced during liver injury is fibronectin. Therefore we investigated the role of laminin, fibronectin and its receptors in HSC migration.

METHODS

We used mouse HSC treated with PDGF‐BB and used Northern and Western analysis to study the expression of dystroglycan (DG) and α5‐integrin (α5‐Int), the respective receptors for laminin (LN) and fibronectin (FN). We also used confocal microscopy to investigate the cellular localization of DG and α5‐Int.

RESULTS

PDGF‐BB induced the cell surface colocalization of α5‐Int and FN as well as DG and LN. While DG and LN were localized to the projections of the HSC, FN and α5‐Int were present along the body of the cells. PDGF‐BB also induced significant changes in the expression and regulation of DG and α5‐Int. While the expression of α5‐Int was up‐regulated that of DG and LN was down‐regulated. However, DG RNA levels were sustained due to the increased transcription of the gene with a 50% decrease in its mRNA t1/2. Antibodies to DG or α5‐Int prevented PDGF‐BB–induced HSC migration.

CONCLUSION

Overall, our findings suggest that both LN and FN are required for HSC migration. However, while the cells migrate on a FN carpet, they appear to be anchoring on LN.
Identification of GRASP65‐interacting proteins and characterization of their roles in Golgi cisternal stacking
Authors: Tang Danming , Wang Yanzhuang
Published: June 2021

In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans‐oligomers. Expression of non‐regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. Further experiments indicate that GRASP65 function is regulated by proteins in the cytosol. In this study, we have applied biochemical approaches to identify GRASP65‐interacting proteins. HeLa cell cytosol was subjected to fractionations using standard biochemical methods followed by affinity chromatography. Proteins that bound to GRASP65 were identified by mass spectrometry and further characterized. Our study has identified potential candidates that play essential roles in Golgi stack formation.
Insight into the Multidrug‐Efflux Pump AcrB Oligomerization Process
Authors: Yu Linliang , Lu Wei , Zhong Meng , Wei Yinan
Published: June 2021

AcrA‐AcrB‐TolC is a three component efflux pump of Escherichia coli. It allows the extrusion of a wide range of cytotoxic compounds. AcrB is located in the inner membrane of the bacterium. It is a homotrimer and utilizes the proton gradient across the inner membrane to drive the export of drugs. In this study, we tested the hypothesis that the intertwining loop of AcrB (residues 211–240) is critical for the assembly of AcrB trimer. Expression and activity assay of AcrB were conducted in an E. coli strain deficient in acrB (ΔacrB). Site‐directed mutagensis studies of the loop region revealed that several residues in the loop were critical for AcrB function. To compare the conformation of the loop in the wild type and mutant AcrB, a cysteine‐pair was introduced in the middle of the loop. The extent of disulfide bond formation was used as a reporter to probe structural changes in the function‐less mutants. Finally, the oligomeric state of the purified mutated protein was determined using blue native polyacrylamide gel electrophoresis (BN‐PAGE). Our results suggest that the loop of AcrB assumed a partially folded, somewhat rigid structure before the trimerization step of protein biogenesis. The source of research support: University of Kentucky faculty start‐up fund to Wei Y. and Kentucky Opportunity Fellowship.
Cooperativity and allosteric regulation of yeast diacylglycerol acyltransferase, DGA1
Authors: Oelkers Peter , Jain Shilpa
Published: June 2021

The esterification of the Kennedy Pathway intermediate diacylglycerol generates the energy storage molecule, triglyceride. Alternatively, diacylglycerol can be used as a precursor for phospholipids. Therefore, diacylglycerol acyltransferase (DGAT), mainly mediated by DGA1 in S. cerevisiae, is a putative control point for the storage or structural utilization of acyl chains. Our in vitro DGAT assays using lro1are1are2 yeast microsomes indicated a distinct sigmoidal relationship between oleoyl‐CoA concentration and relative specific activity (Hill number = 3.0). This apparent cooperativity lead to our hypothesis that DGA1 is allosterically regulated. Subsequent in vitro DGAT assays included metabolites that, based on the regulation of other key enzymes in metabolism, are “metabolic indicators”. Elevated levels of ATP, NADH, acetyl‐CoA, and malonyl‐CoA are indicative of an energy‐rich cell. Elevated levels of AMP, ADP, and NAD+ are indicative of an energy‐poor cell. Initial experiments (n = 3) with estimated physiological concentrations of these metabolites found only 0.8 mM NAD+ to have an effect. The K0.5 was increased by about 20%. Such a direct connection between NAD+ levels and triglyceride synthetic enzymes has not, to our knowledge, been previously reported. Funding was provided by the Pennsylvania ‐ Delaware affiliate of the American Heart Association.
Hsc70 Regulates the Formation of Fusion‐complex Required for VTV‐Golgi Fusion
Authors: Siddiqi Shadab A. , Nafi‐valencia Erika , Rahim Abdul , Siddiqi Shaila , Tiwari Samata , Sabblah Thywill
Published: June 2021

The rate‐limiting step in the secretion of very low‐density lipoproteins (VLDL) from the liver into the plasma is their export from the endoplasmic reticulum (ER) to the Golgi, which is mediated by a specialized ER‐derived VLDL transport vesicle (VTV). VTV fuses with hepatic cis‐Golgi to facilitate the targeted delivery of VLDL to the Golgi lumen. VTV‐Golgi fusion utilizes a specific fusion‐complex composed of a unique set of SNARE proteins. Formation of fusion‐complex requires cytosol, however, cytosolic factors regulating fusion‐complex assembly remain to be identified. To identify cytosolic factors, we performed a series of chromatographic steps including FPLC on liver cytosol and recognized a protein, which we show is required for the formation of fusion‐complex. MALDI‐TOF coupled with tandam mass spectrometry analyses identified this protein as hsc70. Western blotting data using specific antibodies to hsc70 confirmed its identity. Immunodepletion of hsc70 from cytosol resulted in significant (~80%) reduction of fusion‐complex formation. Addition of recombinant hsc70 to the depleted‐cytosol completely restored the activity. Consistent with hsc70 role in fusion‐complex assembly, immunodepletion of hsc70 from hepatic cytosol significantly inhibited VTV‐Golgi fusion. We conclude that hsc70 in hepatic cytosol regulates the formation of fusion‐complex and thus VLDL‐delivery to the Golgi lumen.
New Evidence for a Novel Biosynthetic Pathway to Plasmalogens in Anaerobic Bacteria
Authors: Goldfine Howard , Guan Ziqiang , Johnston Norah C. , Aygun‐Sunar Semra , Daldal Fevzi , Raetz Christian R.H.
Published: June 2021

Study objective

A study of the polar lipids of Clostridium novyi NT and C. tetani was undertaken as part of an ongoing exploration of plasmalogen biosynthesis in anaerobic bacteria.

Approach and results

Our study of the polar lipids of two species of clostridia by thin‐layer chromatography and mass spectrometry has revealed the presence of phosphatidylethanolamine (PtdEtn), cardiolipin and phosphatidylglycerol (PtdGro), as major phospholipids with small amounts of lysyl‐PtdGro and alanyl‐PtdGro in C. novyi. PtdEtn, PtdGro and aminoacyl‐PtdGro were present in both the diacyl and alk‐1′‐enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk‐1′‐enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP‐diacylglycerol and phosphatidylserine were present almost exclusively as diacyl phospholipids.

Conclusions

These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in clostridia. This novel pathway begins with acylation of glycerol‐3‐P and contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis from acyl‐dihydroxyacetone‐P and the alk‐1‐enyl bond is formed by an aerobic mechanism. Research support: GM‐069338, DOE‐ER‐20052, and the University of Pennsylvania.
Structure‐function investigation of PI(4)P binding proteins
Authors: Scott Jordan Leslie , He Ju , Kutateladze Tatiana , Stahelin Robert
Published: June 2021

ADP‐ribosylation factor 1 (Arf1) and four phosphate adaptor protein (FAPP1) are known to co‐localize at the trans‐golgi network and be involved in the process of membrane traffic from the golgi to the plasma membrane. Arf1is a 21 kDa protein known to act in the recruitment of coat proteins and the coating of cargo vesicles by interacting with coatomer and clathrin‐adaptor complex proteins. FAPP1, a 34 kDa protein harboring a phosphotidyl‐4‐phosphate (PI4P) binding PH domain has recently been shown to alter membrane shape in a PI4P‐dependent manner. Though crystal structures are available for both individual proteins, their structural and biophysical interaction has not been elucidated. In collaboration with the Kutateladze lab we have investigated the structural interaction between FAPP1 and Arf1 using X‐ray, NMR, and biophysical analysis. Results demonstrate a high affinity interaction between the active form of Arf 1 and the PH domain of FAPP1. The specificity of the Arf1‐FAPP1 interaction as well as an expansion of PI(4)P‐binding proteins in general will be presented.
Supersizing, volumetrics, or constant eating: causes of increased energy intake in US adults
Authors: Duffey Kiyah J. , Popkin Barry M.
Published: June 2021

The relative effect of energy density (ED), the number of eating occasions (EO), and portion size (PS) to total energy (TE) intake are unknown. Using cross‐sectional nationally‐representative data from the Nationwide Food Consumption Survey (1977–78, n=17,464), Continuing Survey of Food Intake of Individuals (1989–91, n=8,340), and National Health and Nutrition Examination Surveys (1994–98 and 2003–06, n=9,460) for adults (aged ≥ 19 y) we mathematically decompose TE (kcal/d) to understand the relative contributions of each component ‐ PS (g/EO), ED (kcal/g/EO) and EO(#) ‐ to changes in TE over time. There was a steady increase in TE intake (+437 kcal, p<0.001) and the number of daily EO (+1.0 p<0.001) between 1977–78 and 2003–06. The average PS steadily increased between 1977–78 and 1994–98 then non‐significantly decreased between 1994–98 and 2003–06, while the average ED remained relatively stable over the 30‐year period. Between 1977–78 and 1989–91 changes in PS dominated (84.9% of the TE change), while changes in EO accounted for 37.1% of TE change. However, between 1994–98 and 2003–06 changes in EOs accounted for 88.2% and changes in PS accounted for just 22.6% of change in TE. While all three components contributed to the 30‐yr change, changes in EO and PS have consistently accounted for the greatest percent of change in total energy intake. These findings suggest a new focus for obesity prevention efforts.
Topography of Lipid A Assembly and Modification in the Inner Membrane of Francisella
Authors: Zhao Jinshi , Raetz Christian R. H.
Published: June 2021

Francisella tularensis is Gram‐negative bacteria that cause tularemia in mammals. The outer leaflet of its outer membrane is formed by lipid A, the hydrophobic membrane anchor of lipopolysaccharide (LPS). However, about two thirds of the lipid A molecules in Francisella do not have a core oligosaccharide. We have discovered that LPS and free lipid A are not fully separated from each other during purification. We took advantage of a mutation in a mannosyl transferase (LpcC), which adds the proximal mannose residue to Kdo2‐lipid A in the first step of outer core assembly. The LpcC mutant produces a truncated LPS with Kdo residues as the only remaining core sugars. The Kdo modified lipid A molecules lack of both the 1 and 4′‐phosphate groups, whereas free lipid A only lacks the 4′‐phosphate residue. This difference is attributed to the selectivity of the 1‐phosphatase LpxE, which located on the outer surface of the inner membrane and is dependent upon the presence of the Kdo disaccharide for activity. Likewise, LpcC, which faces the cytoplasm, is selective in vivo and in vitro for acceptor substrates with two Kdo units instead of one. Our findings have implications for the order and topography of lipid A modification reactions in the Francisella inner membrane. Supported by NIH grant GM‐51796 to C.R.H.R.
Curcumin induces apoptosis by suppressing protein phosphatase 5 leading to activation of JNK cascade
Authors: Huang Shile , Han Xiuzhen , Xu Baoshan
Published: June 2021

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Our recent studies indicate that curcumin induces apoptosis of cancer cells. This study was set to investigate the underlying mechanism. Here we show that treatment of human cancer cells (Rh30 and HT29) with curcumin activated JNK cascade. This is evidenced by the findings that curcumin increased phosphoryaltion of MKK4 (an upstream kinase of JNK), JNK and c‐Jun (a substrate of JNK). Inhibition of JNK with SP600125 partially prevented curcumin‐induced apoptosis. Similarly, overexpression of dominant negative c‐Jun or downregulation of c‐Jun also in part attenuated curcumin‐induced cell death. Furthermore, we found that curcumin inhibited the activity of protein phosphatase 5 (PP5), a negative regulator of JNK pathway. Overexpression of PP5 blocked curcumin‐induced phosphorylation of c‐Jun and apoptosis. The results suggest that curcumin induced apoptosis through inhibition of PP5, resulting in activation of JNK pathway.
Role of Liver Circadian Rhythm in Adaptive Immunity
Authors: Kashtan Laura Noreen , Menio Jade E , Wheeler Michael D
Published: June 2021

Circadian rhythms are 24‐hour biological cycles that coordinate sleep‐wake cycles, hormone secretion, body temperature, blood pressure, and liver metabolism. Disruption of circadian gene expression has been implicated in changes in metabolism, sleep:wakefulness, and immunity. Our study explores the role of circadian rhythm in the adaptive immune response. To address the hypothesis that circadian genes control adaptive immunity, wildtype mice were housed either in 12:12 light dark cyles (LD) or in complete darkness (DD) for 28 days to disrupt normal circadian cycles. Mice were then challenged with ConA (15mg/kg, intraperitoneal) at CT16 (16 hours post the onset of running bouts) and sacrificed 9 hours after ConA exposure. Serum ALT, innate cytokines (TNF, TGF) and T helper 1 cytokines (gINF) expression were significantly increased by ConA in wildtype mice housed in LD but blunted in mice housed in DD. However, T helper 2 (IL4, IL13) were significantly increased in DD housed mice compared to LD housed mice. Mice deficient in circadian regulator per2 were also resistant to ConA induced liver injury. These data suggest that adaptive immunity is indeed regulated by circadian control. Ongoing experiments will address the precise role of circadian genes such as per2 in the regulation of adaptive immune cells. This work was supported by NIAAA 1R15AA019559.
Distinct, Gene Specific Effect of Heat Shock on Heat Shock Factor‐1 Recruitment and Expression of CXC Chemokine Genes
Authors: Tulapurkar Mohan E , Maity Tapan K , Henry Michael M , Shah Nirav G , Hasday Jeffrey D , Singh Ishwar S
Published: June 2021

Earlier studies have shown that the stress‐activated transcription factor, heat shock factor‐1 (HSF1), modified expression of certain non‐HSP genes including cytokine and chemokine genes. An analysis of human and mouse CXC chemokine genes showed that 28 of the 29 genes had multiple putative heat shock response elements (HSEs) present in their promoters. To determine whether these potential HSEs were functional, we analyzed HSF1 recruitment and expression of 5 human CXC chemokine genes (CXCL‐1, 2, 3, 5 and 8) in heat shocked human lung epithelial‐like A549 cells. Chromatin immunoprecipitation (ChIP) analysis showed that HSF1 was recruited to all but one of these genes (CXCL‐3) and heat shock caused a significant increase in recruitment of HSF1 to one or multiple HSEs present in the promoters of CXCL‐1, 2, 5 and 8 genes. However, the effect of HS exposure on expression of these genes showed a variable gene‐specific effect. For example, CXCL8 expression was markedly enhanced (p<0.05) but CXCL5 expression was significantly repressed (p<0.05) in heat shocked A549 cells. In contrast, expression of CXCL1 and CXCL2, despite HSF1 recruitment to their promoters, was not affected by HS exposure. Our results indicate that some, if not all, putative HSEs present in the CXC chemokine gene promoters are functional and recruit HSF1 in vivo but the effects on gene expression are variable and gene specific.
SIRT1 activators promote p65 deacetylation and suppress TNFa stimulated NF‐kB activation
Authors: Yang Hongying , Feldser Heidi Galonek , Zhang Wei , Zhong Zhong , Loh Christine , Ellis Jim
Published: June 2021

NF‐κB is a transcription factor that mediates immune response and inflammation. The transcriptional activity of NF‐κB is regulated by inhibitor or coactivator proteins as well as posttranslational modifications such as phosphorylation and acetylation. SIRT1, a NAD+‐dependent protein deacetylase, has been shown to deacetylate NF‐κB and suppress stimuli‐induced NF‐κB activation, which opens up a new avenue for treating inflammation through targeting of SIRT1. Our objective in this study was to evaluate SIRT1 and SIRT1 activators in regulating NF‐κB. We have developed a sensitive cellular assay in HEK293 cells to monitor acetylated p65. Consistent with previous reports, overexpression or inhibition of Sirt1 diminishes or escalates the levels of acetylated p65 respectively in these cells. In addition, we show that treatment of the cells with small molecule SIRT1 activators reduces acetylated p65 levels. Furthermore, the compound‐mediated drop in acetylated p65 can be partially reversed by knockdown or inhibition of SIRT1, indicating that the compound effects are dependent on SIRT1. We further demonstrate that these small molecule SIRT1 activators suppress transcriptional activity of NF‐κB induced by TNFα. In conclusion, our data show that small molecule SIRT1 activators mediate deacetylation of NF‐κB and downregulation of its transcription activity, further highlighting the potential use of SIRT1 activators to treat inflammatory diseases.
Effects of nicotine and glucose on production of inflammatory mediators in response to IL‐1 and LPS in human gingival fibroblasts (HGF)
Authors: Sanchez Sharlenne , Chambers Mariah , Madani Mansoor , Borghaei Ruth
Published: June 2021

Periodontitis is the most common cause of adult tooth loss in the U.S., with an estimated 1 in 3 adults suffering from some form and 10 to 15% of adults developing severe forms. In addition to its direct impact, periodontitis also contributes to the development of several other diseases, including cardiovascular disease, pre‐term low birth weight, diabetes and oral cancer Since smoking and diabetes are frequently co‐existing, modifiable risk factors for the development of periodontitis, as well as for cardiovascular disease and some types of cancer, this study is designed to determine whether and to what extent these factors interact to affect the production of proteins and products that are involved in the pathogenesis of these conditions. HGF cultures derived from patients with periodontitis were incubated with IL‐1, LPS, nicotine and glucose in various combinations for 24 hours, and the levels of matrix metalloproteinase 1 and 3 in conditioned media were measured by ELISA. Results show that while nicotine alone has no effect on MMP‐3 protein expression, it acts synergistically with IL‐1 or LPS to increase its expression further. Glucose alone also has no effect on basal or IL‐1 induced MMP‐3 expression but counteracts the effects of nicotine. Similar effects were seen with MMP‐1. Further experiments are planned to determine the effects of nicotine and glucose on these and other mediators of inflammation.
Examination of NAK‐associated protein‐1 homo and hetero‐interactions
Authors: Call Richard Jason , Bell J. Ellis , Bell Jessica K.
Published: June 2021

Protein‐protein interactions form the communication link from membrane‐bound receptors to their downstream cellular targets.

Objective

Our long‐term goal is to examine the protein‐protein interactions responsible for the activation of the Toll‐like receptor 3 (TLR3) signaling cascade that leads to type I interferon production. In the current study, we have targeted the kinase scaffold protein, NAK‐associated protein 1 (NAP1).

Methods

CFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 kinase binding domain (KBD) and scaffold binding motif (SBM) of the kinases, TANK Binding Kinase‐1 (TBK‐1) and IκB kinase epsilon (IKKε), were generated to assess interactions using fluorescence resonance energy transfer (FRET). Biochemical characterization of recombinant targets was completed using gel filtration (GF).

Results

By gel filtration studies, NAP1 KBD elutes as a dimeric species. NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis studies to identify the residues 1ecessary for the kinase recognition sequence are ongoing.

Conclusions

NAP1 KBD forms stable homo‐oligomers and directly interacts with TBK1 and IKKε. Funding was provided by the American Cancer Society.
E‐cadherin antagonizes TGFbeta1 gene induction in hepatic stellate cells by inhibiting RhoA‐dependent Smad3/2 phosphorylation
Authors: Kim Sang Geon , Cho Je , Kim Young Woo
Published: June 2021

Cadherins mediate cell‐cell adhesion and catenin (ctn)‐related signaling pathways. Liver fibrosis is accompanied by the loss of E‐cadherin that promotes the process of epithelial mesenchymal transition. This study investigated whether E‐cadherin overexpression inhibits transforming growth factor‐beta1 (TGFbeta1) gene induction and, if so, what molecular basis. Forced expression of E‐cadherin decreased the level of mesenchymal markers, and the constitutive or inducible expression of the TGFbeta1 gene and its downstream genes. E‐cadherin overexpression decreased Smad3/2 phosphorylations, and consequent its transcriptional activity. The ability of E‐cadherin to inhibit TGFbeta1‐inducible Smad activity was reversed by RhoA activation. Molecular approaches revealed that the p120‐ctn binding domain of E‐cadherin was responsible for TGFbeta1 repression. E‐cadherin was capable of binding p120‐ctn, which recruited RhoA, preventing the ability of TGFbeta1 to increase RhoA‐mediated Smad3 phosphorylation. In the liver tissues of patients with liver fibrosis, E‐cadherin expression reciprocally correlated with the severity of fibrosis. In conclusion, E‐cadherin inhibits Smad3/2 phosphorylations by recruiting RhoA to p120‐ctn, supporting the notion that the loss of E‐cadherin due to cadherin switch promotes TGFbeta1 and target gene induction, and facilitates liver fibrosis.
Vitamin D suppresses staphylococcus mediated inflammation by inhibiting NR4A2 activity in human mesenchymal stem cells
Authors: Maiti Aparna , Jiranek William
Published: June 2021

Mesenchymal stem cells (MSCs) are multipotent cells found in many tissues of the body, could differentiate into stromal tissue such as bone. Staphylococci, particularly Staphylococcus aureus are the predominant cause of bone disease worldwide. In our previous stududies we observed that S. aureus as TLR ligands augmented nuclear factor kB (p65‐NF‐kappaB), pro‐inflammatory cytokines IL‐6, IL8, and NR4A2, a nuclear receptor is emerging as key regulator in disease like prolonged inflammatory responses in MSCs which leads to reduced osteoblast differentiation and increase mesenchymal stem cells proliferations. The active form of Vitamin D [1,25 dihydroxyvitamin D3] has potent immunomodulatory properties which promotes its potential use in prevention of various infectious diseases. Vitamin D mediates its actions by binding to the vitamin D receptor (VDR). This study was aimed to examine the antimicrobial effect of Vitamin D during staphylococcus infection in MSCs. Quantitative realtime PCR data indicated that Vitamin D treatment increased VDR, 1‐alpha hydroxylase, and cathelicidine mRNA levels. Interestingly, Vitamin D treatment along with Staphylococcus aureus infection of MSCs demonstrated significant suppression of NR4A2 expression. Thus Vitamin D is an important contributor to host protection from Staphylococcus infection by inhibiting NR4A2‐mediated inflammation in MSCs.
Exploring regulation in the Toll‐like receptor 3 pathway
Authors: Marion James D. , Roberts Charlotte F. , Bell Jessica K.
Published: June 2021

The innate immune system is the body's first line of defense against infectious agents. Essential to this response are cellular mechanisms that recognize, sequester and eradicate these invading organisms. Toll‐like receptor 3 (TLR3), a pathogen recognition receptor, is stimulated by the viral genomic material double stranded RNA (dsRNA). TLR3 stimulation initiates a signaling cascade that leads to the production of type 1 interferon. Critical to this signaling pathway is a kinase complex, NAP1 (NAK associated protein 1)‐TBK1 (TANK Binding Kinase 1)‐IKKε (IκB kinase epsilon), which leads to the phosphorylation of IRF3 (interferon regulatory factor) and IRF7 and production of IFNβ. To control this activity, SIKE (Suppressor of IKKε) acts as a physiological inhibitor of IKKε and TBK1 activity through an undefined mechanism. Our goal is to define the inhibitory mechanism of SIKE in the kinase reaction. We have examined SIKE for post‐translation modification +/− dsRNA stimulation. We hypothesize that post‐translation modifications may mediate SIKE release from the NAP1‐TBK1‐IKKε complex allowing IRF3 and IRF7 activation. Current studies are focused on defining 1) SIKE post‐translation modifications, 2) how modification may alter SIKE protein interactions, and 3) kinetic analysis of SIKE's effect on TBK1 activity. Funding provided in part by the American Cancer Society.
The role and regulation of protein phosphatase‐1 following oxygen and glucose deprivation in neuroblastoma cells
Authors: Zaydman Anna A , Yu Jieying , Hemmings Hugh C.
Published: June 2021

Protein phosphatase (PP)‐1 is a major serine/threonine phosphatase that dephosphorylates critical regulators of energy metabolism, ionic balance, and apoptosis. PP‐1I, an important regulated form of PP‐1, is activated in vivo following cardiac arrest and resuscitation in a clinically relevant pig model of transient global cerebral ischemia/reperfusion. The role of PP‐1I activation was tested using specific cell‐permeable inhibitors in a neuroblastoma cell (SH‐SY5Y) model of ischemia/reperfusion, oxygen and glucose deprivation (OGD). Activation of PP‐1I purified from pig brain requires pre‐incubation with Mg2+/ATP, consistent with the regulation of PP‐1I activity by an activating kinase. PP‐1I purified from control or ischemic pig brain contained PP‐1 catalytic subunit and inhibitor‐2 (I‐2) regulatory subunit as well as PFTAIRE kinase, a novel protein component of PP‐1I identified by mass spectrometry. We investigated the regulation of PP‐1I phosphatase activity by PFTAIRE kinase using in vitro PP‐1I assays together with phosphorylation assay of I‐2. We also investigated the role of PFTAIRE kinase in PP‐1I activation and cell death following OGD in neuroblastoma cells. These findings implicate PFTAIRE in the ischemic activation of PP‐1I activity in neuronal cells and suggest inhibition of PP‐1I as a possible target for neuroprotection in global cerebral ischemia. Supported by NIH grant NS 56315.
Modulation of the Switch Regions in Ras GTPase
Authors: Holzapfel Genevieve A , Buhrman Gregory , Mattos Carla
Published: June 2021

Ras is a monomeric GTPase which in its GTP‐bound form can bind various effectors in signal transduction pathways. Through its interaction with the effector Raf and associated Ras/Raf/MEK/ERK cascade it is directly involved in the control of cell proliferation and thus implicated in many human cancers. Switch I and switch II undergo conformational transition during hydrolysis and contains active site residues and the effector binding sites. In the Ras/Raf complex only switch I is involved in the interaction. We have a crystal form of Ras in which switch I has the conformation seen in its complex with Raf and switch II is disordered and free of crystal contacts. This facilitated discovery of an allosteric switch, activated in our crystals by calcium acetate, that places the catalytic residue Q61 in the active site. In this crystal form Ras can adopt both the active and inactive states of the allosteric switch, where the activated state is characterized by a shift in helix3/loop7 toward helix 4 and ordering of switch II for catalytic activity. Here we show that DTT stabilizes the inactive form of the switch by occupying a pocket between switch II and helix 3, resulting in an ordered anti‐catalytic conformation of switch II. This anti‐catalytic conformation is also seen in highly transforming mutants, such as RasQ61L. By changing the relative concentrations of calcium acetate and DTT in Ras crystal soaks we show transition between the on and off states of the allosteric switch. Our results provide a proof of concept that the allosteric site and switch II/helix3 pocket can be used to modulate the state of Ras and should therefore serve as novel targets sites in ligand design.

NIH R01 CA096867