bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensitivity to block by penitrem A is reduced in human BK channels containing the β1 subunit
Authors: Asano Shinichi , Dick Gregory M.
Published: June 2021

The ability of antagonists to block large conductance, Ca2+/voltage‐sensitive (BK) channels often depends on whether regulatory β subunits are present. For example, β1 makes BK channels a log order less sensitive to iberiotoxin. Penitrem A is an excellent inhibitor of BK channels; however, whether its effects depend on subunit composition is unknown. We tested the hypothesis that the β1 subunit renders BK channels less sensitive to block by penitrem A. We performed patch clamp recordings on HEK 293 cells transfected with hSlo α or α + β1. Whole‐cell currents were effectively blocked by penitrem A (97 ± 1%). Importantly, however, currents in cells expressing α + β1 subunits were significantly less sensitive to penitrem A (IC50 7 vs. 107 nM; p<0.05). Single channel recordings demonstrated that penitrem A inhibited BK channels in cell‐attached, outside‐out, and inside‐out patches. This membrane permeability of penitrem A is a major advantage over iberiotoxin and tetraethylammonium, as these blockers can inhibit BK only from the extracellular side. As for specificity, penitrem A had no effect on current in cells transfected with KV1.5. Taken together, our data indicate that penitrem A possesses unique pharmacological properties and may be an excellent tool to study BK channels smooth muscle, as these cells generally express β1. (Supported by 5 T32 HL090610)
Involvement of CYP4A‐mediated MAPK pathway in vascular contraction in A2A adenosine receptor knockout mice
Authors: Ponnoth Dovenia S , Nayeem Mohammed A , Kunduri Swati S , Fil Daniel , Tilley Stephen , Ledent Catherine , Mustafa S Jamal
Published: June 2021

We have shown earlier that A2A adenosine receptor (AR) knockout (A2AKO) mice have increased vascular contraction (Ponnoth et al, AJP 2009). We hypothesize that in A2AKO mice, vascular contraction depends on ω‐hydroxylase (CYP4A), through up‐regulation of A1AR via a MAPK pathway. Organ bath and western blot experiments were done with isolated aortas from A2AKO and wild type (WT) mice. A2AKO mice had higher contraction to selective A1 agonist, CCPA (49.2±8.5 vs 27±5.9% at 10−6M, p<0.01) and 20‐HETE (ω‐hydroxylation product; 21.1 ±3.3 vs. 50.6±8.8%). Contraction to NECA (non‐selective adenosine analog) in A2AKO aorta was abolished with p42/p44 MAPK inhibitor PD‐98059 (10−6M). PKC blocker chelerythrine chloride lowered contraction in A2AKO tissues to CCPA (52±8 vs 29±5.2 % at 10−7M, p<0.05). Western blot data indicated higher expressions of A1AR (68 vs. 49%), CYP4A (81 vs 62%), PKC‐ε (69 vs 58%), PKC‐α (59 vs 48%) and p‐ERK 1/2 (92 vs. 66%) in KO tissues than WT. CCPA (10−6M) further increased the expression of these proteins in KO aorta than WT. HET0016 (CYP4A inhibitor; 10−5M) decreased the CCPA‐induced increase in protein expression of A1AR (52%), CYP4A (77%), p‐ERK 1/2 (69%), PKC‐α (45%) and PKC‐ε (32%) in A2AKO tissue. These data suggest that CYP4A mediates contraction in A2A KO mice aorta through up‐regulation of A1AR, possibly through MAPK pathway via PKC isoforms. (HL027339, HL09444, HL071802)
BKCa channel β1 subunit deficiency exaggerates microcirculatory dysfunction, organ damage and mortality in septic mice
Authors: Xu Hui , Wang Youping , Garver Hannah , Fink Gregory G , Galligan James J
Published: June 2021

BKCa channels are composed of pore forming α‐subunits and accessory β1‐subunits that modulate Ca2+ sensitivity of α‐subunits. BKCa channels regulate arterial tone, and earlier studies in human and animals suggested that activation of BKCa channels may contribute to reduced arterial reactivity to norepinephrine (NE) in septic shock. We measured mean arterial pressure (MAP) and heart rate (HR) in lipopolysaccharide (LPS)‐induced endotoxemia in BKCa channel β1‐subunit knockout (KO) mice using radiotelemetry. After LPS administration (20 mg/kg, i.p), KO mice exhibited a more rapid fall in HR and MAP, when compared to wild‐type (WT) mice. There was a shorter latency to mortality and higher mortality rate in KO compared to WT mice. 24 h after LPS administration, mesenteric arteries (MA) from WT and KO mice in vitro demonstrated diminished constrictions to NE and paxilline (a BKCa blocker), but not to ATP. Lung and liver tissues from KO mice had higher myeloperoxidase activity and polymorphonuclear leukocyte infiltration. KO mice demonstrated more severe alveolar collapse, higher wet/dry lung weight, and more severe small intestinal mucosal necrosis. Plasma TNF‐α and IL‐6 levels in KO mice were similar to WT at 6 h post LPS, but these values were significantly higher 24 h post LPS. KO mice did not have higher iNOS expression in MA, lung, liver and intestinal tissues than WT mice. Exaggerated mortality in KO mice also occurred in the cecal ligation/puncture model of septic shock. The results indicate that inhibition of BKCa channels does not protect against hypotension in septic shock. BKCa channel deficiency exaggerates microcirculatory dysfunction, multiple organ damage and mortality via blood pressure‐independent mechanisms.
Brain Microvascular Endothelial Cell Hyperpermeability Following Traumatic Burn Injury: Role of Caspase‐3 Activation
Authors: Tharakan Binu , Whaley J. Greg , Sawant Devendra A , Hunter Felicia A , Childs Ed W
Published: June 2021

Traumatic burn injury is associated with extravasation of fluid from the intravascular space to the interstitium, abdominal cavity and also leads to blood brain barrier (BBB) disruption and brain edema. We hypothesized that caspase‐3 activation following burn would lead to the proleolytic cleavage of the tight junction associated proteins (TJPs) resulting in BBB hyperpermeability and brain edema. Our objective was to determine if burn injury‐mediated rat brain microvascular endothelial cell (RBMEC) hyperpermeability and cleavage of the TJPs, are caspase‐3 dependent. Male Sprague‐Dawley rats were divided into sham and experimental (40% TBSA scald burn) groups. RBMEC were exposed to sham/burn serum and permeability was assessed with FITC‐dextran.TJP integrity was studied using ZO‐1 and occludin immunofluorescnece. Mitochondrial potential (MTP) was determined using JC‐1 and caspase‐3 activity using a fluorometric assay. Burn serum significantly increased permeability compared to sham (p<0.05), and this effect was prevented by the caspase‐3 inhibitor, Z‐DEVD‐FMK (p<0.05). Burn serum treatment significantly decreased MTP and increased caspase‐3 activity (p<0.05). Burn serum disrupted ZO‐1 and occludin that was prevented by Z‐DEVD‐FMK. These results suggest that burn‐mediated blood brain barrier disruption is mediated by caspase‐3 activation and subsequent cleavage of ZO‐1 and occludin.
tPA Contributes To Impairment of ATP and Ca Sensitive K Channel Mediated Cerebrovasodilation After Hypoxia/Ischemia Through Upregulation of ERK MAPK
Authors: Armstead William , Riley John , Cines Douglas , Higazi Abd
Published: June 2021

The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, tPA potentiates impairment of pial artery dilation (PAD) in response to hypotension after hypoxia/ischemia (H/I) in pigs. ATP and Ca sensitive K channels (Katp and Kca) are important regulators of cerebrovascular tone and mediate PAD in response to hypotension. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is upregulated after H/I, with ERK contributing to vasodilator impairment. This study examined the effect of H/I on Katp and Kca PAD and the roles of tPA and ERK in this effect in piglets equipped with a closed cranial window. H/I blunted PAD induced by the Katp agonists cromakalim, calcitonin gene related peptide (CGRP) and the Kca agonist NS 1619, which was exacerbated by tPA. EEIIMD, a hexapeptide derived from plasminogen activator‐1, and ERK antagonist U 0126 prevented Katp and Kca channel agonist PAD impairment while the inactive analogue EEIIMR had no effect. ERK was upregulated after H/I, which was potentiated by tPA. These data indicate that H/I impairs K channel mediated cerebrovasodilation. tPA augments loss of K channel function after injury by upregulating ERK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can produce cerebrohemodynamic dysregulation through impairment of ionic controls of cerebrovascular tone.
Association between Central Artery Stiffness and Cerebral Perfusion in Deep Subcortical White Matter
Authors: Tarumi Takashi , Shah Furqan , Tanaka Hirofumi , Haley Andreana
Published: June 2021

Background

Central arterial stiffness is a strong independent risk factor for cerebrovascular disease. Animal study demonstrated that higher pulse pressure in cerebral artery is associated with hypertrophic remodeling of the microvasculature, leading to increased vascular resistance to the blood flow. Cerebral hypoperfusion is considered the primary cause of cerebrovascular disease including stroke, vascular dementia, and Alzheimer's disease. The aim of this study was to determine whether central arterial stiffness is associated with cerebral perfusion in human.

Methods

A total of 35 middle‐aged adults (15 men and 20 women; 49±7 yrs) were studied. Central aortic stiffness was determined by carotid‐femoral pulse wave velocity. With quantitative perfusion image technique using functional MRI, regional cerebral perfusion in deep subcortical white and gray matter was measured.

Results

Carotid pulse pressure and carotid‐femoral pulse wave velocity were significantly associated with the perfusion in the frontal white matter (r = −0.36 and −0.61 respectively, P<0.05). Hierarchical linear regression analysis revealed that carotid‐femoral pulse wave velocity significantly explains 22 % of variability in the frontal white matter perfusion (β = −0.67, P<0.05), independently from age and blood pressure.

Conclusions

Central aortic stiffness is strongly associated with the cerebral perfusion in frontal white matter even after the adjustment for age and blood pressure.
Intravascular Pressure Augments Cerebral Arterial Constriction by Inducing Voltage‐Insensitive Ca2+ Waves
Authors: Mufti Rania E. , Brett Suzanne E. , Tran Cam Ha , El‐Rahman Rasha Abd , Anfinogenova Yana , El‐Yazbi Ahmed , Cole William C. , Jones Peter P. , Chen Wayne SW , Welsh Donald G.
Published: June 2021

This study examined whether elevated intravascular pressure stimulates asynchronous Ca2+ waves and if their generation contributes to myogenic tone development. Rat cerebral arteries were mounted in an arteriograph, pressurized (20–100 mmHg) and examined under a variety of experimental conditions. Diameter and membrane potential (VM) were monitored using conventional techniques; Ca2+ wave generation and myosin light chain (MLC20)/MYPT1 phosphorylation were assessed by confocal microscopy and western blot analysis, respectively. Elevating intravascular pressure increased the proportion of smooth muscle cells firing Ca2+ waves as well as event frequency. Ca2+ wave augmentation occurred primarily at lower intravascular pressures and ryanodine, an agent that depletes the sarcoplasmic reticulum (SR), eliminated these events. Ca2+ wave generation was voltage‐insensitive as Ca2+ channel blockade and perturbations in extracellular [K+] had little effect on measured parameters. Ryanodine‐induced inhibition of Ca2+ waves attenuated myogenic tone and MLC20 phosphorylation without altering arterial VM. Thapsigargin also attenuated Ca2+ waves, pressure‐induced constriction and MLC20 phosphorylation. The SR‐driven component of the myogenic response was proportionally greater at lower intravascular pressures and subsequent MYPT1 phosphorylation measures revealed that SR Ca2+ waves facilitate pressure‐induced MLC20 phosphorylation through mechanisms that include myosin light chain phosphatase inhibition. Our findings show that mechanical stimuli augment Ca2+ wave generation and that these transient events facilitate tone development particularly at lower intravascular pressures by providing a proportion of the Ca2+ required to directly control MLC20 phosphorylation.
Cigarette smoke enhances TRPC and CD38 expression in human airway smooth muscle
Authors: Burkholder David , Kiel Alexander , Venkatachalem Sathish , Thompson Michael A , Vassallo Robert , Pabelick Christina M , Prakash Y.S. , Wylam Mark E
Published: June 2021

Smoking contributes to or exacerbates diseases such as asthma and COPD, where airway hyperreactivity is a key feature. Enhanced Ca2+ in airway smooth muscle contributes to airway hyperreactivity. We previously determined that the cytokine TNFα upregulates expression of Ca2+ regulatory molecules such as membrane bound bifunctional enzyme CD38 and Ca2+ influx regulatory TRPC3 in human airway smooth muscle cells (HASM). We hypothesized that cigarette smoke (CS) is an inflammatory stimulus that upregulates Ca2+ regulatory mechanisms in HASM. Western analysis showed increased expression of CD38, TRPC3 and TRPC6 in lungs from mice following 4 week in vivo CS exposure. These proteins were also upregulated in HASM cells incubated in 1 or 2% CS extract (CSE), as well as in HASM samples from patients with COPD (compared to controls). In fura‐2 loaded HASM cells, exposure to CSE enhanced histamine‐mediated Ca2+ mobilization as well as store‐operated calcium entry: effects substantially blunted by TRPC3 siRNAs. Finally, in living lung slices of mice exposed for 4 weeks to CS, ACh‐induced airway contractility was substantially greater, compared to controls. These results indicate that CS upregulates expression and function of Ca2+ regulatory proteins, thus contributing to airway hyperreactivity in smoking related diseases. Supported by the Annenberg Foundation (MEW), HL090595 (CMP), FAMRI (RV, YSP).
Cytoskeletal organization alters both epithelial barrier function and immune responses
Authors: Sidhaye Venkataramana , Chau Eric , D'Alessio Franco , King Landon Stuart
Published: June 2021

Changing microtubule polymerization in primary airway epithelial cells alters paracellular permeability. Treatment with nocodazole, which causes microtubule depolymerization, we see a decrease in paracellular permeability. In addition, when we knockdown AQP5, a membrane water channel, we see a decrease in paracellular permeability along with destabilization of microtubules. Our data suggests that AQP5 has a novel role as a microtubule stabilizing protein. Regulation of AQP5 modulates microtubule organization and dynamically regulates barrier function. Furthermore, our data shows that these changes in paracellular permeability regulate polarized epithelial responses to known stimuli such as EGF, IFNg and Substance P, as well as epithelial ligand expression, which mediates one mechanism by which epithelial cells interact with immune cells. Knocking down AQP5 in primary airway epithelial cells significantly increases the expression of the co‐repressor molecule, B7‐H1, which functions to decrease the immune response. Similar findings occur with treatment of the cells with microtubule destabilizing agents. These data suggest that alterations in cytoskeletal organization effect both how an epithelial cell interacts with a neighboring epithelial cell, as well as how it interacts with the immune system.
Dissecting the Mechanism of Unwinding by Human Mitochondrial DNA Helicase
Authors: Sen Doyel , Patel Smita
Published: June 2021

TWINKLE is the only known helicase at the human mitochondrial DNA (HmtDNA) replication fork. It is encoded by PEO1 gene of chromosome 10. It shares 46% amino acid sequence similarity with the helicase T7gene4(gp4) from bacteriophage. Mutations in PEO1 have been linked to deletions in the mitochondrial DNA causing neuromuscular disorders in human. To understand the diseases better, it is crucial to define the molecular mechanism of HmtDNA replication guided by the proteins involved in it. In this study, we have focused on understanding the kinetics of dsDNA unwinding by TWINKLE using ensemble assay with radiolabeled DNA substrates. We have observed that the characteristic unwinding activity of TWINKLE is markedly different from T7gp4. Our most interesting finding indicates that the morphology of non‐translocating strand plays a major role during unwinding by TWINKLE. This is an important step towards understanding the mechanism of initiation of replication from the D‐loop in the mitochondrial DNA. Our final goal is to understand the mechanism of replication of mitochondrial DNA in vitro which in turn would allow us to define the molecular basis of the diseases in human.
Physical models designed to demonstrate the folding pathway of FiP35 WW domain as predicted by molecular dynamics (MD) simulations using Anton, an MD dedicated supercomputer: 2011 Pingry School S.M.A.R.T. Team Project
Authors: Alfano Andrew Benton , Leffler Abba E , Wriggers Willy , Herman Tim , Epifano Alex , Hickey Jack , LaCosta Will , Li Sydney , Meiring Henry , Temares Daniell , Van Besien Alexa , Zhou Ashley , De Luke , O'Mara Deirdre
Published: June 2021

The Pingry School's 2010–2011 S.M.A.R.T. (Students Modeling A Research Topic) Team worked with Abba E. Leffler and Willy Wriggers of D.E. Shaw Research (DESRES) to explore the phenomenon of protein folding. Researchers at DESRES utilized molecular dynamics (MD) in combination with the Anton supercomputer to generate simulations of the fastest folding WW domain reported to date, FiP35 (Shaw et al. 2010). Anton's ability to predict trajectories of the atoms that constitute a protein molecule resulted in the discovery of FiP35's tendency to follow a sequential, well‐defined folding pathway. The Pingry S.M.A.R.T. Team used these findings as a basis for the design of a series of 3‐dimensional models, created with Jmol and a rapid prototyping 3‐dimensional printer, which collectively describe the protein's tendency to fold from its high‐energy, denatured state to its low energy, folded state (native conformation) according to a predictable pathway. The team also organized a corresponding Proteopedia article with embedded Jmol applets and information on the FiP35 folding pathway. This level of visualization enabled by MD simulations assists in understanding the structural basis of protein folding. The Pingry School S.M.A.R.T. Team coordinates with Tim Herman and Shannon Colton of the Center for Biomolecular Modeling at the Milwaukee School of Engineering, which receives funding from NIH‐NCRR‐SEPA.
Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide
Authors: Stipanuk Martha H. , Ueki Iori , Roman Heather B. , Valli Alessandro , Fieselmann Krista , Lam Jimmy , Peters Rachel , Hirschberger Lawrence L
Published: June 2021

Regulation of body cysteine levels is dependent on the regulation of cysteine dioxygenase (CDO), the first enzyme in the pathway for cysteine oxidation to taurine or pyruvate + sulfate. To gain insight into the physiological function of CDO, we generated CDO+/− mice and crossed them to generate CDO−/−, CDO+/−, and CDO+/+ mice. CDO null mice exhibited increased postnatal mortality, a marked growth deficit, and connective tissue pathology. CDO−/− mice had extremely low plasma and tissue taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO‐dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO−/− mice than in CDO+/− or CDO+/+ mice and tissue levels of acid‐labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfuration pathways in the null mice. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO−/− mice. Because H2S has been identified as an important gaseous signaling molecule, pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S levels and facilitate the use of H2S as a signaling molecule. Supported by NIH grant DK056649..
The Quantitative Significance of the Two Transsulfuration Enzymes for Tissue H2S Production
Authors: Xie Peter , Kabil Omer , Vitvitsky Victor , Banerjee Ruma
Published: June 2021

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in various physiological processes such as regulation of blood pressure. Cystathionine beta‐synthase (CBS) and gamma‐cystathioninase (CSE) are the two enzymes that are responsible for the formation of H2S in the transsulfuration pathway. Quantitative Western blot analysis was used to determine the relative abundance of each enzyme in murine liver and kidney. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H2S generation in these tissues and in brain. Our results show that the level of CBS protein is significantly lower than CSE; 60‐fold and 20‐fold in liver and kidney respectively. Levels of both enzymes are higher in liver compared to kidney, 2‐fold and 6‐fold for CBS and CSE respectively. At high substrate concentrations (20 mM each cysteine and homocysteine), CBS and CSE contribute equally to H2S production in liver extract while CBS constitutes the major source of H2S in kidney and brain with CSE contributing approximately 20% and 6% respectively. Determination of tissue levels of these enzymes is important for therapeutic reasons that aim to specifically modulate H2S synthesis in tissue‐specific manner.
Biochemical analysis of free methionine‐R‐sulfoxide reductase
Authors: Kwak Geun‐Hee , Bong Seoung Min , Kim Moon‐Jung , Chi Young Min , Kim Hwa‐Young
Published: June 2021

Free methionine‐R‐sulfoxide reductase (fRMsr) is a new type of methionine sulfoxide reductase that catalyzes the reduction of free methionine‐R‐sulfoxide to methionine. Three conserved Cys residues, Cys‐91, Cys‐101 and Cys‐125, have been demonstrated to be involved in the catalysis by Saccharomyces cerevisiae fRMsr, but their specific functions have not been fully established. In this work, we performed in vivo growth complementation experiments using S. cerevisiae cells lacking all three known methionine sulfoxide reductases. Cells containing a C125S construct, in which Cys‐125 in fRMsr was replaced with Ser, did not grow in methionine sulfoxide medium, whereas cells containing C91S, C101S, or C91/101S constructs could grow in this medium. In addition, when assayed with thioredoxin and glutaredoxin reduction systems, the C125S form was inactive, whereas C91S and C101S had 1–2% and 9–10%, respectively, of the activity of the wild‐type fRMsr. These data show that Cys‐125 is the catalytic residue in fRMsr. Furthermore, we performed structural and biochemical analysis of Staphylococcus aureus fRMsr. Our study suggests the catalytic mechanism of fRMsr in which Cys‐125 functions as the catalytic residue and Cys‐91 as the resolving Cys that forms a disulfide bond with Cys‐125. Cys‐101, previously thought to be a catalytic Cys, is a non‐essential residue for catalytic function in S. aureus fRMsr.
Tetrahydrobiopterin‐dependent Alkylglycerol Monooxygenase: From Gene Identification to Novel Insights into Etherlipid Degradation
Authors: Watschinger Katrin , Keller Markus A. , Golderer Georg , Hermann Martin , Hermetter Albin , Werner‐Felmayer Gabriele , Werner Ernst R.
Published: June 2021

Alkylglycerol monooxygenase is the only enzyme able to degrade alkylglycerols by cleaving the ether bond in a tetrahydrobiopterin‐dependent manner yielding a toxic aldehyde which is further oxidized by fatty aldehyde dehydrogenase.

Using candidate gene expression we have recently identified the gene coding for alkylglycerol monooxygenase (Watschinger K. et al, Proc Natl Acad Sci U S A, 2010, 107:13672‐7). Here, by silencing of TMEM195 in murine macrophage RAW 264.7 cells using lentiviral constructs we confirm that this gene is responsible for tetrahydrobiopterin‐dependent alkylglycerol cleavage.

To investigate the metabolic interplay with fatty aldehyde dehydrogenase we transiently expressed fluorescently‐tagged constructs of alkylglycerol monooxygenase with one of the two splice variants of fatty aldehyde dehydrogenase and analyzed the localization by live cell confocal microscopy. While the major splice variant of fatty aldehyde dehydrogenase (V2) is expressed together with alkylglycerol monooxygenase in the endoplasmic reticulum, the second splice variant (V1) was located exclusively to peroxisomes. This allows the conclusion that the V2 variant is responsible for detoxification of the aldehyde produced by alkylglycerol monooxygenase whereas the V1 variant could be responsible for aldehyde elimination originating from other sources.

Supported by the Austrian Science Fund (FWF) P22406.
Evidence for Direct Cross‐talk between AMP‐activated kinase (AMPK) and O‐linked β‐N‐Acetylglucosaminyltransferase (OGT)
Authors: Bullen John W , Balsbaugh Jeremy , Neumann Dietbert , Hardie Grahame , Hunt Donald F , Hart Gerald W
Published: June 2021

Cycling of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc; a Ser/Thr specific post‐translational modification on nucleocytoplasmic proteins) is a nutrient sensing process regulated by O‐GlcNAc transferase (OGT). Activation of AMP‐activated kinase (AMPK), a vital regulator of cellular energy homeostasis, is also a nutrient sensing process. AMPK and OGT share many downstream targets with the net effect to prolong cell viability under metabolic stress, suggesting significant overlap in AMPK and OGT signaling. However, no studies have investigated whether AMPK and OGT are capable of direct cross‐talk with each other. We demonstrate that: 1) The α and γ subunits of AMPK are GlcNAcylated, 2) Pharmacological and glucose deprivation induced activation of AMPK increases GlcNAcylation of the γ1 subunit, 3) global elevation of GlcNAcylated protein inhibits glucose deprivation induced activation of AMPK, 4) AMPK phosphorylates Thr 172 of OGT in vitro, 5) Acute AMPK activation alters OGT's substrate selectivity. All experiments were performed in differentiated C2C12 myotubes. Collectively, these results demonstrate significant cross‐talk between the AMPK and O‐GlcNAc systems, possibly via coordinated signaling between AMPK and OGT. (Supported by NIH grant R01 DK61671 and R01 CA42486. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU).
STARD5 expression and chemoresistance in lung adenocarcinoma cells
Authors: Clark Barbara J , Meier Renate K , Patel Neesha S , Dougherty Susan M , Klinge Carolyn M
Published: June 2021

DNA‐damaging agents that activate the mitochondrial, caspase‐dependent apoptotic pathway are used to treat non‐small cell carcinoma (NSCLC), but intrinsic and acquired chemoresistance presents a significant clinical problem. Disruption of mitochondria membrane permeability and release of pro‐apoptotic factors is an obligatory step for this pathway and increased mitochondrial cholesterol content has been implicated in chemoresistance in hepatocellular carcinoma. We propose that elevated mitochondrial cholesterol represents a common mechanism that contributes to increased drug resistance in other tumor types. The objective of this study was to determine whether the cholesterol transport protein, STARD5, contributes to chemoresistance in lung adenocarcinoma cells. Western blot analysis demonstrates that STARD5 is highly expressed in lung adenocarcinoma cells compared to normal human bronchial epithelial cells (HBEC). Doxorubicin (Dox)‐ induced cell death was measured using MTT cell proliferation assay and the IC50 values for 5 lung adenocarcinoma cell lines were >2.0 μM Dox compared to <1.0 μM Dox for 5 HBEC lines. The Dox‐treated cells had increased caspase 3/7 activity, supporting that the decrease in cell proliferation was due to increased apoptosis. siRNA silencing of STARD5 in the H1792 lung adenocarcinoma cell line resulted in ~60% increased sensitivity to Dox‐induced apoptosis. Together our data indicate an association between the expression of STARD5 and the resistance of NSCLC to Dox. Mitochondria cholesterol content of these cells remains to be determined.
Zfp206: A novel regulator of adipocyte differentiation potential and cell size
Authors: Pekala Phillip H. , Karschner Vesna A. , Metcalf Mark , Murrell Rachel , Houston Melanie , Sauer Katherine , Stanton Larry
Published: June 2021

The 3T3‐L1 differentiation program leading to the adipocyte phenotype is controlled by a cascade of transcription factors, particularly PPARγ and the family of C/EBP isoforms. Superimposed on this framework we have recently identified the transient expression of the embryonic stem (ES) cell transcription factor, Zfp206, proposed to maintain the differentiation potential of ES cells. In the 3T3‐L1 cells, Zfp206 is expressed at confluency and is attenuated by day 3 as expression switches to Zscan10.201, a splice variant lacking the entire zinc‐finger DNA binding domain which we propose functions as a dominant negative. Our hypothesis is that expression of Zfp206 is essential for maintenance of the differentiation potential and that as terminal differentiation is established with the expression of PPARγ, Zfp206 expression is diminished. The transient expression of the splice variant ZScan10.201 insures the effects of Zfp206 are negated. In murine C/EBPβ−/− ES cells that will not differentiate, Zfp206 was observed to be expressed but did not down regulate, consistent with C/EBPβ involvement in control of expression.

Human preadipocytes also expressed Zfp206 and down regulated expression as the cells differentiated to adipocytes similar to the 3T3‐L1 cells. Suppression of Zfp206 expression using shRNA in the 3T3‐L1 cells, resulted in a marked decrease in cell size as well as an early onset of adipogenesis. Thus, Zfp206 appears to determine both cell fate and cell size. Supported in part by NIH DK088666.
Candida krusei mutants to study virulence and Fluconazole resistance
Authors: Scorzoni Liliana , Leite Fernanda Sangalli , Matsumoto Marcelo Teruyuki , Oliveira Haroldo César , Sardi Janaína C. O. , Benaducci Tatiane , Fusco‐Almeida Ana Marisa , Mendes‐Giannini Maria José Soares
Published: June 2021

Candida krusei is intrinsically resistant to Fluconazole and infects mainly patients suffering from hematologic malignancies and other immunocompromised patients. We constructed mutant of Candida krusei using the Agrobacterium tumefaciens transformation technique and various approaches were developed to understand the mechanism of resistance and the virulence marker as biofilm. The Fluconazol susceptibility of the wild and mutant's strains was analyzed by microdilution test according to CLSI documents. The biofilm production and the proteomic profile of the wild type and sensible strains were also evaluated. Changes in the susceptibility of mutants strain to Fluconazol decreased in 6 folds when compared to wild type, the same strains did not produce biofilms. These results indicate that an important virulence factor may be modified. Interestingly the proteomic dates show different expression of protein support the explanation of intrinsically resistance of Candida krusei to Fluconazol. Supported by CAPES, FAPESP, FINEP and PADC‐FCF.
Molecular characteristics and physiological functions of major royal jelly protein 1 oligomer
Authors: TAMURA Shogo , Kono Toru , Omi Masatomo , Nozaki Reo , Yamaguchi Kikuji , Yamaguchi Yoshihisa , Kobayashi Seiichi , Moriyama Takanori
Published: June 2021

Royal jelly (RJ) contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble RJ proteins. In this study, we focused the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using Blue Native (BN)‐PAGE. MRJP 1 oligomer was separated into 55‐ and 5 kDa spots on 2‐dimensional BN/SDS‐PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, while the 5 kDa protein was identified as Apisimin by N‐terminal amino acid sequencing, and this protein may function as a subunit‐joining protein within MRJP 1 oligomer. Furthermore, MRJP 1 oligomer dose‐dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity.
Changes of rat plasma proteome from E15.5 to newborn
Authors: Wei Lilong , Wu Shuzhen , Gao Youhe
Published: June 2021

To investigate the changes of plasma proteome in the process of fetal development, the plasma proteome of Fetal (E15.5) and newborn (NB) rat were profiled using LC‐MS/MS and compared. With a rigorous filter, we found 24 of relative high abundant proteins were only identified in the Fetal (E15.5) rat plasma, while 12 of relative high abundant proteins only in the newborn (NB) rat plasma. Comparing with Fetal (E15.5) rat plasma, 10 of relative abundance proteins decreased in the newborn (NB) rat plasma, while 21 of proteins increased. Some changes are consistent with previous knowledge such as albumin, apolipoproteins, Plasminogen, Corticosteroid‐binding globulin£¬Haptoglobin, Alpha‐2‐macroglobulin, and Alpha‐fetoprotein, etc. With the Gene Ontology analysis, immune and stimulus related proteins were found to be enriched in the changed proteins that we identified. At the same time some organ specific proteins were also included in these changed proteins, such as Prothrombin£¬Fetub protein, and Alpha‐fetoprotein, etc. This is the first study of plasma changes during fetal development with proteomics technology..
Genetic determinants of aerobic degradation of the antidepressant sertraline by Pseudomonas fluorescens Pf‐5
Authors: Palanski Brad A , Schultz Melissa M , Strand Stephanie S , Snider Mark J
Published: June 2021

Pharmaceuticals are now a well‐recognized class of environmental contaminants that have been shown to elicit negative effects on aquatic life at environmentally relevant concentrations. Sertraline (Zoloft™) is an antidepressant that is ubiquitous in aquatic environments near wastewater treatment plants (WWTPs). Recently we reported the isolation of a bacterium (Pseudomonas fluorescens Pf‐5) from a WWTP that co‐metabolizes sertraline with ethanol as a primary carbon source. Here we investigate the biochemical pathway by which catabolism occurs. An extraction technique for sertraline and its catabolites from bacterial liquid cultures was developed, and analysis by liquid chromatography‐mass spectrometry shows that norsertraline and other catabolites are present. Currently, we are utilizing high‐throughput DNA sequencing to characterize the transcriptome when the bacterium is grown in the presence and absence of sertraline. Analysis of differential gene expression should implicate genes involved in sertraline catabolism and facilitate mechanistic studies of enzymes for which the genes code. This study will further our understanding of the function and evolution of bacterial catabolic pathways and will aid in a comprehensive assessment of the environmental effects of sertraline and its catabolites. This work was supported by a grant from HHMI through the Undergraduate Science Education Program.
Effective preparation method of human catalytic antibody light chains
Authors: Uda Taizo , Honjo Eijiro , Hifumi Emi
Published: June 2021

We have successfully produced mouse type catalytic antibody light chains (antigenases) by immunizing ground‐state peptides or proteins. The antigenase possesses the ability to enzymatically decompose a targeting molecule using the catalytic triad formed in the structure. In the case of human antibody light chains, the catalytic triad was mostly encoded in Subgroup II, suggesting that the light chains belonging to the subgroup should exhibit enzymatic functions.

In order to verify the above concept, we amplified an antibody germline gene as the cDNA belonging to Subgroup II, which was taken from human leucocyte. The cDNA was inserted in pET system, followed by a transformation to express the protein (human antibody light chain) in E.coli. We could recover the protein with very high purity by passing two affinity columns. Its catalytic activity was measured using synthetic substrate (peptide‐MCA) to monitor the fluorescence generated by the cleavage of the peptide‐MCA bond, suggesting the human antibody light chain exhibited the amidase activity. Furthermore, the light chains showed other interesting enzymatic reaction in addition to the amidase activity.
Impact of NHE1 Phosphorylation by Ribosomal S‐6 kinase and RhoA kinase on ERM binding and cell motility
Authors: Nguyen Tony , Wallert Mark , Provost Joseph
Published: June 2021

Cellular migration involves complex signaling and intricate reorganization of the cytoskeleton. Ezrin/Radixin/Moesin (ERM) proteins play a crucial role in the reorganization of the cytoskeleton by binding to membrane bound proteins including the Na+‐H+ exchanger (NHE) and anchoring stress fibers to the plasma membrane. During cellular migration, there is a reorganization of the membrane to localize NHE to the leading edge of the cell. Localization of NHE helps to give the cell polarity, which is important in directing stress fiber formation to assist the cell to move forward. The focus of this study was to determine the conditions by which ERM translocates to the membrane to bind to NHE and examine the role of the ERM:NHE binding complex in proper formation of unidirectional cell migration. We have prepared a series of Ser or Thr – Ala mutants at putative RhoA Kinase (Rock) and Ribosomal S‐6 kinase (RSK) phosphorylation sites and expressed these in NHE1‐null PS120 cells. Co‐immunoprecipitation of NHE1 identified ERM‐NHE1 interactions for each mutant following activation with lysophopsphatidic acid (LPA). Using wild‐type and non‐phosphorylatable EFGP‐ERM, we investigated the localization of ERM proteins and the impact of NHE1 phosphorylation. Finally we show the effect of NHE1 and ERM phosphorylation on stress fiber formation and cell motility.. This work was supported with funds from NSF‐MCB‐081778, NSF‐RUI‐MCB 0930432, and NIH‐1‐R15‐CA135616‐01.
Steroidogenic Acute Regulatory (StAR)‐Related Lipid Transfer Domain Protein 5 (STARD5) expression is associated with cholesterol content of human kidney proximal tubule cells
Authors: Clark Barbara J , Meier Renate K , Khundmiri Syed J , Lederer Eleanor D
Published: June 2021

STARD5 belongs to the START protein superfamily of lipid transport proteins and functions in intracellular non‐vesicular cholesterol transport. We have reported that STARD5 expression is localized to proximal tubules of mouse kidney and the objective for this study was to determine the effect of STARD5 on cholesterol efflux. Using the HK‐2 and HKC‐8 human proximal tubule cell lines, we measured 1) expression of STARD5 and the ABCA1 cholesterol efflux pump by qRT‐PCR and Western blot analysis, 2) total free cholesterol content by cholesterol oxidase assay, and 3) efflux of 3[H]‐cholesterol to the ApoA‐1 acceptor protein. Compared to HK‐2 cells, the HKC‐8 cells have 2‐fold greater STARD5 protein and an 80% increase in total free cholesterol levels. Cholesterol efflux was ~2.5‐fold more efficient in HK‐2 cells, most likely due to 3–4‐fold greater ABCA1 expression levels in this cell line. siRNA silencing of STARD5 expression resulted in a 70% increase in cholesterol efflux in HKC‐8 cells but had no effect in HK‐2 cells. Together our results support that higher expression of STARD5 may function to sequester intracellular cholesterol and impede cholesterol efflux in renal proximal tubule cells with limiting ABCA1 expression levels.
Structure of dimeric apoA‐IV: basis for HDL model
Authors: Deng Xiaodi Andy , Davidson W Sean , Thompson Thomas B
Published: June 2021

A major contributing factor to cardiovascular disease is atherosclerosis and its progression is dramatically affected by HDL and LDL levels. HDL and LDL are lipoprotein complexes consisting of lipids and proteins from the apolipoprotein family. Apolipoprotein members have a unique detergent‐like property, which enable them to bind lipids and form large complexes such as chylomicrons, VLDL, LDL and HDL. Another key property of apolipoprotein is self‐association and elucidating this mechanism is key to understanding the functions of cardio‐protective lipoprotein complexes like HDL. The few solved structures of apolipoproteins have failed to provide insight into self‐association. We have determined the first dimeric structure of apoA‐IV, one of the largest of the apolipoproteins at 43kDa. From the structure, we observed a novel dimerization mechanism that buries over 11,000 Å2, forming an incredibly stable dimer that requires over 48 hours at 37°C to reach monomer:dimer equilibrium. This unique mechanism also maintains a four‐helical bundle upon dimerization and creats a hydrophobic core that stretches over 150Å and providing an ideal environment for sequestering lipids. Using this structure in conjunction with biochemical data, we have generated a model of apoA‐IV transitioning from a monomer to a HDL in three steps. The apoA‐IV HDL model is supported by previous observations and rectifies some conflicting biochemical data in the field. This structure not only reveals the mechanism for apolipoprotein self‐association, but also provides a clear picture that demonstrates the apoA‐IV structure as the basis for its biological function. We believe this transition model is a general mechanism and can extend to apoA‐I and apoA‐V, which also form HDL.
C2 Domains: Versatile Lipid Binding Modules
Authors: Stahelin Robert V , Gunasekaran Christopher , Yu Hui
Published: June 2021

The C2 domain (~130 residues) was originally identified as one of two conserved regulatory domains of Ca2+‐dependent protein kinases C. A large number of proteins containing the C2 domain have been identified since, and most of them are involved in signal transduction or membrane trafficking. Early experiments demonstrated C2 domains could bind bulk phospholipids such as phosphatidylserine or phosphatidylcholine with high affinity and specificity, while more recently; C2 domains have been shown to interact with different phosphoinositides and/or sphingolipids in both Ca2+‐dependent and ‐independent manners. C2 domains have also been shown to bind specific proteins, interact with phosphotyrosine, induce membrane curvature changes, participate in intramolecular interactions, and interact with viral proteins.

How the multifaceted C2 domains achieve diverse signaling and interaction properties is only beginning to come to fruition. We have begun a comprehensive and rigorous experimental, computational, single molecule, and structural analysis of the mammalian C2 domain family, which will lead to a better understanding of how these properties manipulate the full‐length proteins' functions.
The Molecular Basis of Ceramide‐1‐Phosphate Recognition by Peripheral Proteins
Authors: Ward Katherine Elizabeth , Sudhahar Christopher G. , Olmstead Deborah N. , Stahelin Robert V.
Published: June 2021

The sphingolipid ceramide‐1‐phosphate (C1P) plays a critical role in the cellular signaling that mediates inflammation, cell proliferation and phagocytosis. C1P has been shown to increase the activity of cytosolic phospholipase A2 α (cPLA2α) as well regulate its translocation to cellular membranes, a process that promotes inflammation through the production of arachidonic acid. In this study we have resolved the first C1P binding site of a peripheral protein. Using NMR we demonstrate the cPLA2α C2 domain interaction site for C1P by mapping chemical shifts. Subsequently, this novel‐binding site is confirmed with in vitro biophysical and biochemical analysis as well as molecular dynamics simulations. To search the human genome for other C1P binding proteins we have performed proteomic studies to began high throughput characterization of the conserved nature of C1P binding.

Funding source: American Heart Association SDG0735350N (R.V.S)
Computational Prediction and Experimental Validation of the Lipid‐Binding Regions of Hsp70 Proteins
Authors: McCallister Chelsea , Donnelly Gabrielle , Le Dat , Nikolaidis Nikolas
Published: June 2021

Hsp70s function in protein folding and refolding, transportation across biological membranes, and are necessary for cellular homeostasis under both physiological and pathological conditions. Apart from their intracellular functions, Hsp70s have also been found in close proximity or bound to biological membranes and in the extracellular milieu. It has been hypothesized that interaction of Hsp70s with specific lipids allows them to translocate and anchor to membranes, and subsequently be secreted. However, these hypotheses have not been directly tested, since the lipid‐binding regions (LBRs) of Hsp70s and the amino acids that mediate these interactions remain uncharacterized. To identify the LBRs of the Hsp70s, we have developed a new algorithm, because the existing protein‐domain‐identification algorithms fail to predict any known lipid‐binding domains in them. Application of our algorithm on HspA1A predicted two regions as putative LBRs. We tested these predictions experimentally and determined that both regions bind to lipids with high affinity. Therefore the experimental results support the predictions of our computational strategy and define two regions that mediate lipid‐binding on the HspA1A. We plan to expand the use of our algorithm to test the evolutionary conservation or divergence of the lipid‐binding function of Hsp70s using Hsp70s from evolutionarily key species.

This project was supported by funds from CSUPERB and CSUF to NN
Antioxidant and anti‐inflammatory effects of lutein in aorta and liver of guinea pigs fed with a hypercholesterolemic diet.
Authors: Kim Jung Eun , Lee Ji‐Young , Fernandez Maria‐Luz
Published: June 2021

The effects of the carotenoid, lutein on atherosclerosis and liver oxidation were tested in guinea pigs fed a hypercholesterolemic diet (0.25 g/100 g dietary cholesterol). Guinea pigs were divided into two groups, control group (n = 9) or Lutein group (n = 10) (0.1 g/100 g lutein) and fed the experimental diets for 12 wk. Hematoxylin and eosin (H&E) stained aorta was examined and aortic and liver malondialdehyde (MDA) concentrations were analyzed by TBARS assay. Plasma and aortic cytokine concentrations were measured by Immunoassay and liver enzymes, alkaline phosphatase (ALP) and alanine transaminase (ALT) were measured. Aortic MDA concentrations were lower in the Lutein (1.7 ± 1.3 nmol/mg protein) compared to the control group (3.0 ± 1.4 nmol/mg protein) (P < 0.05) and H&E staining indicated separation and disrupted morphology of aortas in control guinea pigs while these features were not shown in the Lutein group. Aortic MDA positively correlated with aortic cytokines. Plasma concentration of ALP and ALT were not different between groups, however, liver MDA concentrations were lower in the Lutein group (6.0 ± 1.0 nmol/mg protein) compared to the control group (7.0 ± 1.0 nmol/mg protein) (P < 0.05). Liver MDA concentrations were negatively correlated with liver lutein concentrations (r=−0.469, p<0.05). These results suggest protective antioxidant and anti‐inflammatory effects of lutein in both liver and aorta.