bluuebunny/crossref_metadata_2025 huggingface dataset:
Arrestin‐3 binding to JNK1alpha1/JNK2alpha2: modulation of JNK1 and JNK2 activity via scaffolding
Authors: Kook Seunghyi , Zhan Xuanzhi , Kaoud Tamer S , Dalby Kevin N , Gurevich Vsevolod V , Gurevich Eugenia V
Published: June 2021

Non‐visual arrestins scaffold mitogen‐activated protein kinase (MAPK) cascades, bringing together three components of MAPK signaling modules. The c‐Jun N‐terminal kinases (JNKs) are members of MAPK family and three JNKs isoforms (JNK1, JNK2, and JNK3) have been identified. JNK1 and JNK2 are ubiquitously expressed, whereas JNK3 is expressed mainly in testes and neuronal tissues. Arrestin‐3 isoform has been shown to enhance JNK3 activity by scaffolding JNK3 with upstream kinases. However, it has not been documented whether arrestin‐3 can modulate the activity of JNK1 and JNK2 isoforms. Using purified proteins we found that arrestin‐3 directly binds JNK1α1 and JNK2α2. Reconstitution of JNK1α1 or JNK2α2 signaling module with either MKK4 or MKK7 in the presence of different arrestin‐3 concentrations showed that arrestin‐3 acts as scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation at optimal concentration. Arrestin‐3 also enhanced phosphorylation of endogenous JNK1 and 2 isoforms when upstream kinases MKK4 or MKK7 were overexpressed together with arrestin‐3 in COS‐7 cells. Thus, the data suggest that arrestin‐3 also modulates JNK1 and JNK2 activity in non‐neuronal cells acting as a scaffold for these JNK isoforms and upstream kinases.

Support: NIH Grants NS065868 and DA030103 (EVG), GM077561, GM081756, and EY011500 (VVG), GM059802 and Welch Foundation Grant F‐1390 (KND).
Overcoming Drug‐Induced Resistance in BRaf Mutated Melanoma Cells
Authors: Samadani Ramin , Jung Kwan‐Young , Zhang Jun , MacKerell Alexander , Fletcher Steven , Shapiro Paul
Published: June 2021

Melanoma is the fastest growing cancer in the United States with metastatic melanoma patients having a median lifespan of less than one year. Most melanomas are characterized by an upregulation of the extracellular signal‐regulated kinase (ERK1/2) pathway with more than 80% of patients harboring a constitutively active mutation in BRaf. New treatments inhibiting BRaf or its direct targets MEK1/2 have recently been developed and have shown promising results. Regrettably, these results are invariably short lived with resistance to the new therapeutic agent quickly taking hold. The objective of this study is to determine if a novel class of thiazolidinedione compounds designed to target the ERK1/2 proteins can overcome some of the mechanisms of resistance that have been identified. Specifically, our studies indicate that the thiazolidinedione compounds can overcome resistance induced by mutations that occur in Ras as well as overexpression of MAP3K8 found in drug resistant melanoma cells. These resistance pathways are important clinically because they render current BRaf and MEK targeted therapies ineffective. It is believed that these thiazolidinedione compounds can be developed into a new class of inhibitors aimed at helping patients overcome resistance to current treatments.
Molecular basis for a bipartite phosphatase interaction with the anchoring protein AKAP79
Authors: Nygren Patrick J , Gold Matthew G , Weisbrod Chad R , Bruce James E , Scott John D
Published: June 2021

The calcium/calmodulin‐activated protein phosphatase 2B (PP2B, or calcineurin) regulates diverse biological processes including glucose homeostasis and synaptic plasticity. PP2B is targeted to specific substrates by scaffolding molecules such as A‐kinase anchoring protein 79 (AKAP79), which also interacts with protein kinase A (PKA). Previous work from our lab shows the stoichiometry of this complex to be 2 AKAP79:4 PP2B:4 PKA and identified a dimerization domain on AKAP79. AKAP79 contains a PxIxIT motif near the C‐terminus that is a primary site of interaction with PP2B. We have identified an additional interaction site for PP2B at the N‐terminal region of AKAP79. Using hybrid approaches including chemical crosslinking, native mass spectrometry, and in vitro pulldown assays, we show that this additional interaction exclusively binds activated PP2B through a conserved LxVP mechanism. We identify a 16 amino acid region in AKAP79 that is necessary for the LxVP interaction and show that the peptide can effectively compete for binding. These findings contribute to a dynamic model of PP2B anchoring in which the active phosphatase is anchored differently then the inactive form. Research supported by NIH grant GM48231.
Structural basis of stable PP2A latency by α4‐protein
Authors: Stanevich Vitali , Jiang Li , Sengupta Rituparna , Satyshur Kenneth , Watkins Guy , Wadzinski Brian , Kong Mei , Xing Yongna
Published: June 2021

Phosphoprotein α4 is essential for cell survival and is known as an inhibitor of protein phosphatase 2A (PP2A). α4 binds to PP2A catalytic subunit (PP2Ac) and stabilizes it in an inactive, latent form. Strikingly, we show that α4 binds preferentially to the partially folded PP2Ac. The crystal structure of α4 bound to a folding sub‐domain of PP2Ac (PP2AFD) reveals that α4 binding entails a distorted PP2A active site and displacement of an internal packing near the phosphatase active site. Measurement of the H‐D exchange of the α4‐bound PP2Ac versus active enzyme reveals conformational changes in PP2Ac associated with α4 binding that correlates with the crystal structure. Our structural observations imply that binding of α4 to the inner surface of PP2Ac may serve dual function: 1) establish PP2A latency; 2) stabilize PP2Ac by preventing aggregation of the partially folded or newly synthesized PP2Ac and protecting PP2Ac from proteasomal degradation by shielding a PP2A polyubiquitination site. In addition, disruption of PP2A‐α4 interaction in vivo is sufficient to cause cell death and complete loss of PP2Ac in cells. Our data reveal molecular details of α4‐PP2A interaction and offer structural mechanism of α4 function and stable PP2A latency.
Structural insights into tight control of PP2A methylation and function by LCMT‐1
Authors: Stanevich Vitali , Jiang Li , Satyshur Kenneth , Li Yongfeng , Jeffrey Philip , Li Zhu , Menden Patrick , Semmelhack Martin , Xing Yongna
Published: June 2021

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase in all eukaryotic cells, which functions as holoenzyme consisting of catalytic (PP2Ac), regulatory and scaffolding subunits. Formation of PP2A holoenzymes is regulated by post‐translational modifications of the C‐terminus of PP2Ac (C‐tail), most notably carboxyl methylation on Leu‐309 catalyzed by leucine carboxyl methyl transferase 1 (LCMT‐1). LCMT‐1 is crucial for cell survival: its knockdown reduces the level of PP2A methylation and induces apoptosis in mammalian cells. We determined the crystal structure of the PP2Ac‐LCMT‐1 complex, trapped by covalent crosslinking of PP2A carboxyl terminus with a chemical mimic of SAM, at 2.7 Å. The structure suggests that methylation needs correct positioning of C‐tail, which, in turn, requires a direct contact of LCMT‐1 with the PP2A active site in addition to its interaction with C‐tail. To confirm structural observations, we measured dependence of PP2A methylation on the conformation of its active site. As expected, removal of catalytic Mn2+ ions led to the simultaneous elimination of PP2A phosphatase and LCMT‐1 methyltransferase activities. On contrary, stimulation of PP2A phosphatase activity by PP2A phosphatase activator (PTPA) led to stimulation of PP2A methylation. Altogether, data provide molecular basis for PP2A methylation that is tightly coupled to activation of PP2A phosphatase activity.
Src Kinase Mediates Renal Interstitial Fibroblast Activation and Proliferation
Authors: Ma Li , Tang Jinhua , Ponnusamy Murugavel , Zhuang Shougang
Published: June 2021

Renal Interstitial fibroblast activation and proliferation is central to the development and progression of renal fibrosis after various insults, however, the signaling mechanism regulating this process is incompletely clear. In this study, we examined the role of Src, a non‐receptor tyrosine kinase in renal interstitial fibroblast activation and proliferation. Exposure of cultured renal interstitial fibroblasts (NRK‐49F) to PP1, a specific Src inhibitor, resulted in decreased expression of alpha‐Smooth muscle actin and fibronectin, two hallmarks of fibroblast activation and type 1 collagen, a key extracellular matrix protein, in a dose‐dependent manner. Silencing Src with siRNA also significantly inhibited the expression of these three proteins. Moreover, inhibition of Src kinase activity with either PP1 or siRNA, also blocked cell proliferation, decreased expression of cyclin D1, cyclin B1 and cyclin E and increased expression of p27 and p21. Finally, we showed that STAT3, AKT and ERK 1/2 were phosphorylated in cultured NRK‐49 cells and presence of PP1 or knockdown of Src with siRNA inhibited phosphorylation of all of them. Collectively, our results reveal an important role of Src in mediating activation and proliferation of renal interstitial fibroblasts and suggest that Src inhibition might be a potential therapeutic approach for attenuating renal fibrogenesis.
Protein kinase C mutations in cancer
Authors: Antal Corina E. , Kang Emily , Newton Alexandra C.
Published: June 2021

Protein kinase C (PKC) levels are aberrant in diverse cancers and over 160 mutations in PKC have been identified in human cancers. Here we characterized PKC mutations identified in human cancers to investigate whether PKCs function as oncogenes or tumor suppressors or whether their function is isozyme and/or context dependent. Live‐cell imaging was used to determine how these mutations affect PKC translocation, subcellular localization, and activity. Cell‐based assays were used to determine how PKC mutations alter proliferation, apoptosis, migration, and anchorageindependent growth. Of 30 mutations we examined thus far, 14 of them reduced or completely abolished PKC activity, while only 1 increased it. These mutations reside within the diacylglycerol‐binding C1 domain, the Ca2+‐binding C2 domain, or the kinase domain, within both conventional and novel PKCs. The inactivating mutations ablate activity by either impeding Ca2+ or diacylglycerol binding, or by yielding a misfolded kinase. These results suggest that PKC can act as a tumor suppressor. Understanding whether mutations are activating or inactivating in specific cancers would allow us to determine whether therapeutic strategies should target ways to inactivate or activate PKC in the respective cancers.

This work was supported by GM43154 to ACN and DGE1144086 and T32 GM007752 to CEA.
Using ProPeL to discover the binding specificity of human DYRK1a
Authors: Lubner Joshua , Chou Michael F. , Church George M. , Schwartz Daniel
Published: June 2021

Pairing kinases to their substrates is central to elucidating signal transduction pathways and understanding their functions in normal and diseased states. Kinases discriminate between potential substrates in part by linear sequence “motifs” surrounding the residue to be phosphorylated, yet motifs for the majority of the 518 human kinases remain unknown. One largely uncharacterized kinase is Dual specificity tyrosine‐phosphorylation‐regulated kinase 1a (DYRK1a), which is implicated in Down's Syndrome, Alzheimer's Disease, alternative splicing and synaptic trafficking. Using our new Proteomic Peptide Library (ProPeL) methodology, in which we express a human kinase in E. coli and analyze the resulting phosphopeptides with our pLogo and motif‐x software, we extracted a strong sequence motif for DYRK1a from 933 unique phosphorylation sites. We also detected 8 novel DYRK1a autophosphorylation sites. Currently, the only known tyrosine targets of DYRK1a are on DYRK1a itself. Strikingly, however, we observed 66 unique phosphotyrosine sites on substrates other than DYRK1a, suggesting that its ability to phosphorylate tyrosine residues may not be limited to autophosphorylation. Finally we predicted human DYRK1a substrates using the authors’ scan‐x tool. This study was funded in part by grants from the University of Connecticut Research Foundation (DS) and Genomes to Life from US DoE (GMC).
Structure of the eukaryotic atypical RIO kinases reveals ATPase‐dependent mechanism in ribosome biogenesis
Authors: LaRonde Nicole A.
Published: June 2021

RIO kinases are ancient atypical protein kinases essential for the final maturation step of the small subunit of the ribosome. The crystal structures of two eukaryotic RIO kinases, Rio2 from Chaetomium thermophilum and human RioK1, in the presence of ATP, were determined to 2.5 and 2.8 Å respectively. Unexpectedly, a novel phosphoaspartate intermediate was observed in both structures. Acyl‐phosphate intermediates, observed in P‐type ATPases, are unusual for serine protein kinases. Prompted by this new information, we show that RIO kinases have significant ATPase activity. Additionally, docking of the eukaryotic Rio2 kinase into a reported cryo‐electron microscopy map of the pre‐40S particles and yeast genetic studies show that although catalytic activity is required for function, the active site is inaccessible when the RIO domain is bound to the pre‐ribosome. A new model for the functional mechanism of the RIO kinases is therefore suggested that includes a role for a transient intermediate and ATPase activity in pre‐40S maturation. We propose that RIO kinases function not as protein kinases, but as ATPases that sense pre‐40S conformational change and drive particles to maturation. These data collectively provide surprising new insight into the role of RIO kinases in ribosome biogenesis.
Investigations into the role of Lantibiotic Cyclase‐like (LanCL) proteins in mammals
Authors: Zeng Min , Chen Jie , van der Donk Wilfred
Published: June 2021

Lantibiotics are a group of antibiotics that are ribosomally synthesized and posttranslationally modified peptides. Lantibiotic cyclase (LanC) catalyzes the Michael addition reaction of the thiol group in cysteines to the α, β‐unsaturated carbonyl in dehydrated amino acids in the precursor peptides, generating the macrocyclic structures of lantibiotics.

Interestingly, in eukaryotes, which do not produce lantibiotics, homologs of LanC widely exist. LanC‐like (LanCL) proteins have highly conserved sequences as well as the Zn2+ ligands and active site residues that are also found in LanC. However, the function of these proteins and the underlying mechanisms are largely unknown. In the current study, we discovered a novel function of LanCL2 in the regulation of Akt phosphorylation and insulin signaling in mammalian cells and propose a possible mechanism of this regulation. Our discoveries suggest the functional significance of LanC‐like proteins in mammals.
The identification, characterization, and subcellular localization of a novel potential GTPase activating protein involved in cytokinesis
Authors: Faitar Silviu Leonard , Kilijanski Jordan J , Heassler Kyle W , Davie Jeremiah J
Published: June 2021

Rab proteins constitute the largest class of the Ras superfamily of monomeric guanine nucleotide‐binding (G) proteins, with over 60 members identified so far. They function as molecular switches, cycling between an active GTP‐bound and an inactive GDP‐bound state, and controlling all the major steps in membrane trafficking. Their activity is regulated by guanine nucleotide exchange factors (GEFs), which mediate their activation, and by GTPase‐activating proteins (GAPs), which terminate their activity. Recent studies suggested that Evi5 is a potent GAP for Rab11, and has a critical role in the late stages of mitosis. Bioinformatics analyses revealed a new Evi5‐like protein (Evi5L) possessing a putative Rab‐GAP domain similar to Evi5. In addition, Evi5 and Evi5L conservation among the Eukarya was evaluated. Fluorescence microscopy was used for the subcellular localization of Evi5L in both interphase and mitotic Hep G2 cells. Furthermore, immunocytochemical studies confirmed that Evi5 and Evi5L have similar dynamic localizations during the last stages of mitosis, and suggested that both proteins may be involved in targeting different subpopulations of endocytic vesicles to the midbody during cytokinesis.
Over‐expression of Mutant Rheb Proteins and mTOR Signaling
Authors: Teran Daniel , Parmar Nitika
Published: June 2021

Activation of mTOR is regulated by the small G‐protein, Rheb. In mammalian systems there are two Rheb genes‐Rheb1 and Rheb2 (RhebL1). Based on a scan for putative residues subject to phosphorylation, we created several mutants for both Rheb1 and Rheb2 using a site‐directed mutagenesis protocol. These residues include Ser130, Lys45, Thr20, Ser44, and Gln130. Wild type and mutant Rheb proteins were over‐expressed in HEK293 cells and impact on mTOR signaling was assessed via monitoring the phosphorylation of the S6 protein. Experiments were also conducted under serum starved conditions to determine if over‐expression of wild type and/or mutant Rheb proteins offered a growth advantage to the cells. Phospho‐S6 was analyzed via immunoblotting in cells grown in enriched, starved and starved/stimulated conditions. Our preliminary results indicate that over‐expression of both wild‐type Rheb1 and Rheb2 is able to rescue the starved phenotype observed, i.e. decrease in S6 phosphorylation, however, the mutants S44A and K45A were unable to rescue this phenotype. Variations in phospho‐S6 observed for other mutants suggest that they may allow the cells to continue growth under starved conditions, similar to wild‐type. Our results so far are unique and provide evidence that the residues S44 and K45 may play a critical role in regulating Rheb's activity, thus warranting a deeper investigation of Rheb's impact on mTOR signaling.
Automated analysis of Ca2+ mobilization in the cornea under hypoxia
Authors: Lee Albert , Derricks Kelsey , Trinkaus‐Randall Vickery
Published: June 2021

Previously, our lab demonstrated that ATP release upon injury induces Ca2+ mobilization in corneal epithelial cells via purinergic signaling. This mobilization was shown to be critical for the promotion of corneal wound repair. Most recently, we demonstrated that glutamatergic and purinergic receptors modulate communication between corneal epithelia and its innervating neurons by eliciting a complex bimodal Ca2+ wave upon injury. In this study, our goal was to examine changes in Ca2+ mobilization caused by hypoxia during corneal wound repair.

Ca2+ mobilization was analyzed by an algorithm using MATLAB. When epithelial cells were treated with neuronal wound media, hypoxia decreased both Ca2+ mobilization and subsequent cluster formation. There was a reduction in the amount of ATP present in both primary trigeminal rat neurons and human corneal limbal epithelial cells (HCLEs). Hypoxia reduced response to agonists specific to P2Y by 45% and to P2X receptors by 64%. Wound closure was impaired and the phosphorylation of paxillin was decreased.

Our results demonstrated that hypoxia impairs cell‐cell communication upon injury, which correlated with changes in the purinergic signaling pathway. Our automated Ca2+ mobilization analysis showed a reduction in nucleotide‐induced Ca2+ mobilization and a decrease in cluster formation under hypoxia.
A Role for the Excess Ryanodine Receptors in Excitation‐Contraction Coupling
Authors: Pitake Saumitra , Ochs Raymond S.
Published: June 2021

A close interaction between the plasma membrane bound DHPR (dihydropyridine receptor, the sensor of electrical changes in the membrane) and the endoplasmic reticulum bound RyR (ryanodine receptor, the Ca2+ release channel to the cytosol) is central to excitation‐contraction coupled Ca2+ release (ECC) by skeletal muscle. We hypothesized that the 2:1 ratio of RyR:DHPR may reflect relative movement of DHPR and RyR accompanied by bursts of Ca2+ release. We tested the hypothesis by measuring the sensitivity of the RyR and DHPR to modulators of these proteins. The response of RyR (measured as Ca2+ appearance in the cytosol) to its namesake modulator ryanodine (Ry) was shifted to lower concentrations of Ry when cells were subjected to field depolarization. In a similar way, Ca2+ release in response to the DHPR activator BayK was also shifted to lower concentrations of BayK when cells were depolarized. However, sensitivity of Ca2+ release to these agents was not altered when either the direct RyR activator CMC (a caffeine‐like agent) or increased medium Ca2+ was applied. While some studies have suggested that RyR may not be essential in ECC, this was based on very high concentrations of Ry (above 100 μM). Yet, we found that cells incubated with Ry at 10 μM completely blocked ECC. Ry at concentrations greater than 50 μM compromised cell viability. Our findings support an interaction model of DHPR and RyR in ECC, a role for the excess number of RyR over DHPR, and indicate Ca2+ release for ECC is exclusively through the RyR.
Nasal mucus contains cAMP and cGMP which are critical factors in preservation of normal olfaction
Authors: Henkin Robert I
Published: June 2021

Olfaction is a sensory modality comprised of a tripartite complex of physiological components – brain, nerves and receptors. However, the majority of pathological changes which inhibit olfaction relates not to changes in nerves or brain but to changes in presence and character of olfactory receptors. Olfactory receptors are unique organs. They do not possess blood vessels or lymphatics, do not exhibit mitosis and are constantly being remodeled. All growth and development of olfactory receptors depend upon stem cells which are stimulated by growth factors to supply the chemical moieties responsible for stem cell action. These growth factors are constituents of nasal mucus. Growth factors consist of a variety of chemical moieties which influence stem cell function and thereby olfactory receptor growth and development. Growth factors are funneled from multiple body organ systems into nasal mucus where they act to stimulate stem cells. cAMP and cGMP are major growth factors in nasal mucus. Inhibition of these or other growth factors in nasal mucus will inhibit olfactory function. X‐irradiation to the nasal cavity related to tumor treatment inhibits total nasal mucus production and thereby produces smell loss (hyposmia) by inhibiting growth factor secretion. Inhibition of cAMP, cGMP or any other growth factor component of nasal mucus such as carbonic anhydrase VI or thyroxine also inhibits stem cell stimulation of receptor growth and development. Olfactory end organs respond to these stimuli to create the complex cellular growth patterns which result in normal olfactory cellular development and thereby normal olfaction. Interference with secretion or synthesis of cAMP and cGMP in nasal mucus causes hyposmia whereas activation of secretion of these cyclases corrects hyposmia.
ATP Inhibition Couples Guanylyl Cyclase A and B to Cellular Energy Status
Authors: Potter Lincoln Ross , Mauseth Matthew Allen , Robinson Jerid Wayne
Published: June 2021

Homodimeric and heterodimeric guanylyl cyclases (GCs) regulate numerous biologic functions by catalyzing the synthesis of cGMP in response to peptide or gaseous ligands, respectively. Atrial natriuretic peptide (NP) and C‐type NP reduce the Km for GC‐A and GC‐B, respectively, by a process that requires ATP binding to a high affinity allosteric activation site in the catalytic domain with an EC50 of ~0.1 mM. Here, we show that higher physiologic concentrations of ATP inhibit GC‐A and GC‐B by binding a low affinity site with an IC50 of ~2 mM in the catalytic domain. Unlike activation, inhibition neither required the 2'ribosyl OH group of ATP nor CNP. The inhibition was mixed type and was observed with ATP and ADP but not AMP. Inhibition was competitive with the product, pyrophosphate but not the substrate, GTP. We suggest that changes in cellular ATP concentrations couple GC activity to the energy status of the cell by binding the pyrophosphate site.
The Effect of Phosphatidylinositol 4,5‐bisphosphate Depletion on the Internalization of G Protein‐coupled Receptors
Authors: Tóth Dániel J , Tóth József T , Tallósy Bernadett , Hunyady László , Várnai Péter
Published: June 2021

Phosphatidylinositol 4,5‐bisphosphate (PtdInsP2) has been shown to be critical for many endocytic processes including the internalization of G protein‐coupled receptors (GPCRs). Our aim in this study was to compare the effect of different plasma membrane PtdInsP2 depletion methods on GPCR internalization.

We used bioluminescence resonance energy transfer (BRET) to follow the internalization of the luciferase‐tagged β2 adrenergic receptor (β2AR) in HEK 293 cells. To reduce PtdInsP2 levels, we applied either the rapamycin‐inducible recruitment of a 5‐phosphatase domain to the plasma membrane, or a truncated form of type 1 angiotensin receptor (AT1R) which activates phospholipase Cβ. We determined the rate of PtdInsP2 degradation using the PH domain of phospholipase Cδ1 which binds PtdInsP2 specifically, and found it to be comparable for the two depletion methods. While PtdInsP2 depletion by our rapamycin‐based system inhibited the internalization of β2AR, PtdInsP2 depletion by AT1R had no effect on it, measured by the same method.

Our data suggest that the effect of plasma membrane PtdInsP2 depletion on the internalization of β2AR can be different depending on the method by which the lipid is degraded. Further investigation is needed to determine whether this discrepancy is due to degradation of distinct PtdInsP2 pools of the plasma membrane or other factors are responsible for it.

Support: OTKA K105006
Endomembrane HRas controls the PI3 kinase/Akt/eNOS signaling cascade in VEGF induced endothelial cell migration
Authors: Haeussler Dagmar , Burgoyne Joseph R , Hou Xiuyun , Pimental David R , Cohen Richard A , Bachschmid Markus M
Published: June 2021

We demonstrate for the first time that endomembrane‐delimited HRas mediates VEGF‐induced activation of eNOS and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total HRas is S‐palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane localized HRas unexplained. Knock‐down of HRas blocked VEGF‐induced PI3K‐dependent Akt (S473) and eNOS (S1177) phosphorylation and nitric oxide‐dependent cell migration, demonstrating the essential role of HRas. Activation of endogenous HRas led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous HRas was fully restored by modest overexpression of an endomembrane‐delimited HRas palmitoylation mutant. These studies define a newly recognized endomembrane localization of HRas‐mediated nitric oxide‐dependent proangiogenic signaling.
A muscle‐specific isoform of neuronal nitric oxide synthase is phosphorylated in response to insulin
Authors: Hinchee‐Rodriguez Kathryn , Garg Neha , Venkatakrishnan Priya , Roman Madeline G. , Adamo Martin , Masters Bettie Sue , Roman Linda J.
Published: June 2021

Nitric oxide (NO), a signaling molecule made by nitric oxide synthases (NOSs), is implicated in skeletal muscle glucose uptake and insulin sensitivity. In skeletal muscle from adults with Type 2 Diabetes (T2DM), insulin‐stimulated NO production and glucose disposal is decreased. Additionally, NO production increases in normal adults with insulin, but not in T2DM adults. The primary NOS form in skeletal muscle is nNOSμ, which differs from nNOSα by a 34‐amino acid insert into a known regulatory region. While it is well‐documented that insulin causes endothelial NOS (eNOS) activation via serine phosphorylation in the C‐terminal tail, little is known of nNOSμ function or response to insulin. Because nNOS contains analogous serine residues in its C‐terminus, phosphorylation of nNOS in C2C12 myotubes was examined at 0–60 minutes after 100nM insulin exposure. Insulin treatment resulted in increased phosphorylation of the nNOS variants at early time points (up to 15 min), but decreased at later time points. Additionally, under conditions of free fatty acid (FFA)‐induced insulin resistance, a further increase in phospho‐nNOS levels was observed, suggesting that FFA may have a synergistic effect on nNOS phosphorylation with insulin stimulation. nNOS phosphorylation was also observed in vivo in muscle tissue from insulin‐treated WT C57/Bl6 mice. Supported by NIH GM052419 to LJR and BSM.
Protein phosphatase 2A B56α mediates retinoic acid‐induced decreases in phosphorylation of endothelial nitric oxide synthase at serine 1179 and nitric oxide production
Authors: Jo Inho , Park Jung‐Hyun , Sung Hye Youn , Lee Jee Young , Kim Hyun Jin , Ahn Jung‐Hyuck
Published: June 2021

All‐trans retinoic acid (atRA) decreases nitric oxide (NO) production through Akt‐mediated decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS‐Ser1179) in bovine aortic endothelial cells (BAEC). Since protein phosphatase 2A (PP2A) was also reported to decrease eNOS‐Ser1179phosphorylation, we investigated using BAEC whether PP2A mediates atRA‐induced eNOS‐Ser1179dephosphorylation and subsequent decreased NO production. Treatment with okadaic acid (5 nM), a selective PP2A inhibitor, or ectopic expression of siRNA of PP2A catalytic subunit α (PP2A Cα) significantly increased eNOS‐Ser1179phosphorylation and NO production. Each treatment also significantly reversed atRA‐induced observed effects, suggesting a role for PP2A. We also found that atRA significantly increased cellular PP2A activity. However, western blot analysis revealed that atRA did not increase the expression of PP2A Cα, although it significantly increased the level of B56α of PP2A regulatory B subunit (PP2A B56α) but not PP2A B55α and PP2A B56δ. Real‐time PCR assay confirmed a significant increase in PP2A B56α mRNA expression in atRA‐treated cells. Ectopic expression of siRNA of PP2A B56α significantly reversed atRA‐induced inhibitory effects on eNOS‐Ser1179 phosphorylation and NO production, suggesting a role for PP2A B56α. Our study demonstrates for the first time that atRA decreases eNOS‐Ser1179phosphorylation and NO release at least in part by increasing PP2A B56α‐mediated PP2A activity in BAEC. This study was supported by NRF 2012R1A2A2A01004914 and 2011–0027347.
Probing the Structure of Photoreceptor Phosphodiesterase (PDE6) and Its Interactions with Transducin by Cross‐linking and Molecular Modeling
Authors: Gao Xiong‐Zhuo , Zeng Xiaohui , Zhang Xiu‐Jun , Chu Feixia , Cote Rick H
Published: June 2021

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme of visual signaling. Its activity is regulated by the inhibitory subunit (Pγ). Upon light stimulation, activated transducin (Tα*) relieves Pγ inhibition of the PDE6 catalytic dimer (Pαβ), activates PDE6, and causes a drop in cellular cGMP levels. However, the mechanism by which Tα* activates PDE6 is unclear. Although structures for some PDEs have been solved, no high‐resolution structures of PDE6 are available. Using homology modeling, we generated models of the GAF and catalytic domains of the α and β catalytic subunits. Using PDE2 (PDB:3IBJ) as a template, we built a topology model for the full‐length Pαβ catalytic dimer. Chemical cross‐linking of Pαβ identified 18 cross‐links that validated our homology model. We also identified multiple cross‐links between Pγ and Pαβ that support an extended structure for Pγ bound to Pαβ, as well as sites within the N‐terminal region of Pγ which may allosterically regulate cGMP binding to PDE6 GAF domains. Preliminary cross‐linking results of Tα*‐activated PDE6 reveal multiple sites of interaction of Tα* with Pα, Pβ, and Pγ. We conclude that cross‐linking and molecular modeling provide unique insights into PDE6 regulation not available by other approaches. Supported by NIH grant EY‐05798 (RHC), NH Agricultural Experiment Station grant NH00567 (FC), and the Karabelas Faculty Development Fund (FC).
High blood pressure and arterial stiffness are not associated with low bone mass
Authors: Johnson Sarah A. , Figueroa Arturo , Navaei Negin , Wong Alexei , Kalfon Roy , Elam Marcus , Payton Mark , Arjmandi Bahram H.
Published: June 2021

Hypertension (HTN), arterial stiffness and osteoporosis have common etiologies and their occurrence increases dramatically after menopause. HTN and arterial stiffness have been shown to be inversely associated with bone mass. The objective of this cross‐sectional study was to examine the degree to which bone mineral density (BMD), bone mineral content (BMC) and percent body fat (PBF) are influenced by high blood pressure (BP) and arterial stiffness in 38 postmenopausal women with pre‐ and stage 1‐HTN. Systolic and diastolic BP were evaluated using resting brachial BP and arterial stiffness was assessed using carotid‐femoral pulse wave velocity (cfPWV) and right brachial‐ankle pulse wave velocity (RbaPWV). BMD, BMC and PBF were measured using dual‐energy X‐ray absorptiometry (DXA). Pearson correlation coefficients were used to determine the relationship between variables of interest. Systolic BP, diastolic BP and cfPWV were not correlated with BMD (P = 0.98, 0.55, and 0.63, respectively), BMC (P = 0.77, 0.94, and 0.47, respectively) or PBF (P = 0.44, 0.88, and 0.35, respectively). RbaPWV was not correlated with right leg BMD (P = 0.79) or right leg BMC (P = 0.39). There was a strong negative correlation between RbaPWV and PBF (r = − 0.45, P = 0.004). These findings do not suggest that there is a link between high BP, arterial stiffness and bone mass but do suggest a link between body fat and systemic arterial stiffness.

Grant Funding Source: Internal Funding
The effects of short‐term participation in the Supplemental Nutrition Assistance Program on food security and dietary intake of low‐income Massachusetts adults: a pilot study
Authors: Leung Cindy W , Cluggish Sarah , Villamor Eduardo , Catalano Paul J , Willett Walter C , Rimm Eric B
Published: June 2021

In a pilot study of 107 low‐income Massachusetts adults, we examined if the initiation of Supplemental Nutrition Assistance Program (SNAP) benefits improved food security and dietary behaviors over a three‐month period. New SNAP participants were also asked their opinions on strategies to improve the diets of program participants. On a scale of 0 (high food security) to 10 points (very low food security), SNAP participation was associated with a 0.5‐point (95% CI −1.4, 0.4), nonsignificant improvement in food security, after adjustment for age, sex, marital status, weight and health status. Compared to nonparticipants, SNAP participants had a significant increase in refined grains (1.1 servings/day, 95% CI 0.2, 2.0), and a marginal increase in sweets and bakery desserts (0.4 serving/day, 95% CI −0.4, 1.0) from baseline to follow‐up. No associations were observed with other food groups, total energy, macronutrients, or overall dietary quality. Most SNAP participants agreed that providing incentives to purchase fruits and vegetables (86%), providing more cooking classes or nutrition education (86%) and restricting the purchase of sugar‐sweetened beverages with SNAP benefits (59%) would help SNAP participants to eat better. Although short‐term SNAP participation may marginally improve food security, there were few improvements in dietary intake among this group of low‐income Massachusetts adults.
Food Security and Supplemental Nutrition Assistance Participation Associated with Home Availability and Preschool Child Consumption of 100% Fruit Juice
Authors: Yarbrough Katherine , Schilling Elizabeth A , Ferris Ann M
Published: June 2021

Preschool children consume more than the American Academy of Pediatrics recommendation of 4–6 oz of 100% fruit juice (FJ) per day. This study analyzed the association of food security and Supplemental Nutrition Assistance Program (SNAP) and Women, Infants, and Children (WIC) status with home availability and child consumption of FJ. Caregivers of 3–5 y old children (n=462) provided demographics and child consumption quantity and completed the USDA food security module and a home beverage inventory. Sixty‐one percent of children consumed >;6 oz FJ/d. SNAP status was associated with consumption and food security was associated with availability in non‐parametric analyses; WIC was not associated with either. Controlling for caregiver ethnicity and age, ordinal logistic regressions of FJ availability (5 levels) and consumption (5 levels) on food security and SNAP were estimated. Food secure homes had more FJ available than food insecure homes (p<.05). Children in SNAP homes consumed more FJ than children in non‐SNAP homes (p<.05). In summary, a majority of children consumed more FJ than recommended. Food security and SNAP status are associated with home availability and consumption of FJ, respectively.
Are physical activity and food insecurity associated with depressive feelings in U.S. adults?
Authors: McNeill Meghan , Venci Brittany Jeanne , Park Sohyun , Lee Seung‐Yeon
Published: June 2021

We examined associations between depressive feelings and food security while controlling for physical activity (PA) in a nationally representative sample using the 2011 National Health Interview Survey data for 29,981 U.S. adults (≥18 years). Multivariable logistic regression was used to estimate adjusted odds ratio (OR) and 95% confidence interval (CI) for associations between food security and having depressive feelings after controlling for sociodemographic and lifestyle factors. Food security scale was used to define food secure (score 0–2), low food secure (3–5), and very low food secure (6–10). About 87% were food secure, 6.6% were low food secure and 5.2% were very low food secure. Regardless of PA status, both low and very low food secure adults had higher odds of having each of 6 depressive feelings compared to physically active, food secure adults. For example, very low food secure, physically inactive adults had higher odds for feeling sad (OR=3.40, 95% CI=2.75, 4.20), nervous (OR=2.47, 95% CI=1.98, 3.07), restless/fidgety (OR=3.00, 95% CI=2.36, 3.82), hopeless (OR=3.28, 95% CI=2.62, 4.09), everything was an effort (OR=3.13, 95% CI=2.51, 3.89), and worthless (OR=3.68, 95% CI=2.86, 4.75) than physically active, food secure adults. Although the directionality of our associations is unknown, our findings suggest that food insecure, physically inactive adults tend to have more depressive feelings.

Grant Funding Source: None
Why have NCDs received so little attention in Nigeria?
Authors: Adeyemi Olutayo , Pelletier David L. , Devine Carol M. , Pinstrup‐Andersen Per , Stoltzfus Rebecca J.
Published: June 2021

Noncommunicable diseases (NCDs) in low and middle income countries (LMICs) increase illness, death and poverty, overburden health systems, and may retard progress on undernutrition and infectious diseases through resource competition. Global authorities have urged that “a sound and explicit government policy” is vital for tackling NCDs in LMICs; yet, policy actors’ views concerning NCDs in these countries have not been studied. This research examined the state of NCD policy and the views of policy actors in Nigeria, which is ranked fifth among LMICs in terms of mortality due to NCDs. Methods included document analysis and semi‐structured interviews with 34 key Nigerian policy actors. Interview results were compared with established factors that shape political priority.

The Nigerian health ministry has had a NCD policy unit since 1989 and there has been significant policy rhetoric about NCDs. However, there is no formal policy or serious action on NCDs. Factors contributing to this neglect of NCDs include the biological/social characteristics of NCDs, lack of bureaucratic or political champions, lack of a broadly‐based, effective NCD coalition and, until recently, limited attention by the international community.

Research support: Cornell University: Division of Nutritional Sciences; Mario Einaudi Center for International Studies. First Presbyterian Church in Ithaca International Hunger Student Grant
The components of metabolic syndrome in relation to weight status in South Indians
Authors: Must Aviva , Thanikachalam Mohan , Begum Mubeen , Vijayakumar Harivanza , Chui Ken , Chomitz Virginia , Bermudez Odilia , Thanikachalam Sadagopan
Published: June 2021

Asian populations have elevated risk for obesity‐related morbidity such as metabolic syndrome (MS). MS, defined as the presence of 3 of 5 cardiovascular and anthropometric indicators, may overlook differences in its components relative to modifiable antecedents, such as weight. A population‐based cross‐sectional survey in 8042 (44% M, 56% F) South Indians collected detailed physicals and biochemical assays via standard protocols. MS components were defined based on the modified National Cholesterol Education Program Adult Treatment Panel (ATPIII) criteria for Asians. BMI categories were <18.5, 18.5–23, 23–25, 25–30, and 30+.

11% M and 7% F were underweight (BMI<18.5); prevalence of BMI>;23 was 56% M and 70% F; with 6% M and 16% F with BMI >;30. MS components were prevalent across all weight groups BMI >;18.5, with ranges 27 to 59% M and 18 to 80% F. Compared to normal weight (BMI 18.5–23), after adjusting for smoking (males only) and age, prevalence ratios (PRs) for triglycerides, high density lipoproteins, elevated blood pressure/hypertension, and fasting plasma glucose/diabetes showed patterns of increasing risk across BMI categories, with PRs of 1.3 to 1.7 M and 1.2 to 2.1 F in the BMI 30+ category. Results were similar for waist circumference categories. Overall, the risk gradient is less apparent than observed in western populations, likely due to the high prevalence of these risk factors in normal weight groups.
Animal and plant protein intakes among different regional diets and their association with overweight and obesity
Authors: Zhai Fengying , Wang Huijun , Zhang Bing , Du Shufa
Published: June 2021

Few studies have explored the protein intake‐overweight relationship among different regional diets. Our study assesses dietary animal and plant protein intake in Chinese population with different regional diets and examines the association between food group‐specific derived animal and plant protein intakes and body mass index (BMI) and waist circumference (WC). Data from the China Health and Nutrition Survey covering nine provinces with randomly selected samples with different cooking systems and dietary behaviors were used. Dietary data were collected by using three consecutive 24‐hr dietary recalls. Generalized linear models were used to investigate the association between dietary protein intake and BMI and WC, adjusting for age, gender, education, occupation, demography (urban and rural), regional diet, and energy intake. Protein intake from animal sources was positively associated with BMI (β=0.023, p<0.001) and WC (β=0.079, p<0.001), while dairy‐derived protein was inversely associated with BMI (β= −0.070, p<0.001) and WC (β= −0.235, p<0.001). Legume‐derived protein intakes were positively associated with BMI (β=0.008, p<0.05) and WC (β=0.033, p<0.001). This study suggests that promoting plant sources food and diary consumption in China where milk intake is very low may play a role in protecting against obesity in Chinese adults.
Prospective analysis of the formulation of country‐level plans of a regional health partnership
Authors: Gonzalez Wendy , Frongillo Edward A , Thrasher James F
Published: June 2021

Reducing malnutrition, which contributes to more than one third of all deaths in children under age 5, is currently an urgent global priority. Global Health Partnerships (GHPs) work together with country partners in the formulation of action plans that address pressing health challenges such as malnutrition. Formulation of country action plans is often a contentious process. The successful formulation of an action plan occurs when the process pursues goals of feasibility, alignment, and ownership. We prospectively examined the formulation of two country‐level action plans of a regional health partnership in Latin America, and assessed whether these three goals were pursued. Data were collected using participant observation of key events; semi‐structured, in‐depth interviews with involved actors; and document review. We found that, although a GHP can promote feasibility, ownership, and alignment, the country context is a key determinant of whether these goals can be reached. Lack of nutrition plans and aims, weak leadership of the Ministry of Health, and an upcoming political transition were factors that prevented attainment of these three goals. By recognizing that a complex context can delay or impede the attainment of these goals, GHPs can be responsive to the country‐specific challenges, devise appropriate procedures to address them, and adapt expectations to the context.

Grant Funding Source: None
Preventing the global burden of type 2 diabetes by improving the quality of staple foods: The Global Nutrition and Epidemiologic Transition Initiative
Authors: Mattei Josiemer , Malik Vasanti , Wedick Nicole M , Spiegelman Donna , Hu Frank B , Willett Walter C , Campos Hannia
Published: June 2021

Type 2 diabetes has reached epidemic proportions globally, affecting low‐ and high‐income countries. Two main contributors have been the epidemiological transition and concomitant adverse changes in diet. We aimed to depict the current state of diabetes in light of the ongoing epidemiological and nutrition transition, and to posit that a key factor has been a shift in the type and processing of staple foods, using data from 10 countries participating in the Global Nutrition and Epidemiologic Transition Initiative (GNET), a collaborative effort aiming to reduce diabetes by improving the quality of traditional foods through culturally‐appropriate interventions. We depicted the epidemiological transition using median age, life expectancy, overall death rate and cause of death, and the nutrition transition using data from food balance sheets. Main staple foods (rice, roots, tubers, wheat, corn, legumes) differed by country, with most countries undergoing a shift in principal contributors to energy in the past 60 years. A notable example was the opposing trend in percent energy from rice (positive) vs. legumes (inverse). GNET countries with pilot data have documented barriers and motivators to increase intake of high‐quality traditional foods. In summary, global research efforts to identify and promote intake of culturally‐acceptable high‐quality staple foods could play a key role in diabetes prevention.
Diet and other health‐related behaviors of individuals with impaired glucose tolerance, cardiovascular disease (CVD) or CVD risk: A global view from the NAVIGATOR study
Authors: Bales Connie , Haffner S , Thomas L , Sun J , Yates T , Huffman K , Califf R , Toumilehto J , McMurray J , Holman R , Davies M , Kraus W
Published: June 2021

Type 2 diabetes (T2D) and cardiovascular disease (CVD) are escalating worldwide; yet the practice of health‐promoting life‐style behaviors on a global scale is mostly unstudied. We examined baseline characteristics of 9,013 participants (median age: 63 yr; BMI: 29.7 kg*m‐2) enrolled in the Nateglinide and Valsartan in Impaired Glucose Tolerance Outcomes Research (NAVIGATOR) study; all had impaired glucose tolerance, CVD or CVD risk and were from North America (n = 2146; NA), Latin America (n =1406; LA), Europe (n=4909; EUR) and Asia (n=552; AS). Participants answered 6 questions about diet (inclusion of fruits/vegetables, avoidance of fat, saturated fat, sugar, salt, alcohol), measured steps taken (pedometer), and provided other health measures. The majority (73.4%) reported >;3 prudent diet behaviors; this was linked with being female, older, a non‐smoker, and having a lower BMI and waist circumference. A higher (p<0.0001) number of prudent diet behaviors was reported by AS versus other regions. Rates of smoking were highest (12.7%) and limiting alcohol lowest (79.1%) in EUR and pedometer steps taken were AS>;LA>;EUR>;NA. Self‐rated health was highest in AS and NA. Thus, among a global population of adults with T2D and CVD risk, adherence to healthy diet and exercise behaviors was strong and highest among those in the AS region. Research supported in part by Novartis Pharma.