Critical role for PI3-kinase in regulating the use of proteins as an amino acid source

ABSTRACTDarunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIVDRVRP51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIVDRVRP51are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIVI54Mand rHIVI84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIVV32Ialso developed high-level DRV resistance. However, wild-type HIVNL4-3(rHIVWT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIVWT, rHIVV32Iwas highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIVWTwith DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1’s DRV resistance development and elucidate, at least in part, a mechanism of DRV’s high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution.IMPORTANCEDarunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, allin vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

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Sodium coupled neutral amino acid transporter SNAT2 counteracts cardiogenic pulmonary edema by driving alveolar fluid clearance

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00461.2020 ◽

2021 ◽

Author(s):

Sarah Weidenfeld ◽

Cécile Chupin ◽

Delia Isabel Langner ◽

Tamador Zetoun ◽

Simon Rozowsky ◽

...

Keyword(s):

Amino Acid ◽

Epithelial Cells ◽

Pulmonary Edema ◽

Hydrostatic Stress ◽

Critical Role ◽

Neutral Amino Acid ◽

Lung Edema ◽

Alveolar Fluid Clearance ◽

Pharmacological Inhibition ◽

Pulmonary Epithelial Cells

The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 (SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of L-alanine across pulmonary epithelial cells. Homozygous Slc38a2-/- mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/- mice developed higher wet-to-dry weight ratios (W/D) as compared to WT in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/- mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.

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Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes

Journal of Bacteriology ◽

10.1128/jb.00543-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 4

Author(s):

Germán E. Piñas ◽

John S. Parkinson

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Kinase Activity ◽

Critical Role ◽

Active State ◽

Dynamics Simulation ◽

Cross Linking ◽

Atp Binding ◽

Content Type

ABSTRACT Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, “dipped” and “undipped,” for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units. IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called “dipped” CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

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Lactobacillus frumenti mediates energy production via fatty acid β-oxidation in the liver of early-weaned piglets

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-019-0399-5 ◽

2019 ◽

Vol 10 (1) ◽

Author(s):

Zhichang Wang ◽

Jun Hu ◽

Wenyong Zheng ◽

Tao Yang ◽

Xinkai Wang ◽

...

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Gut Microbiota ◽

Oral Administration ◽

Energy Production ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Critical Role ◽

Guanosine Monophosphate ◽

Weaned Piglets

Abstract Background Early-weaning of piglets is often accompanied by severe disorders, especially diarrhea. The gut microbiota and its metabolites play a critical role in the maintenance of the physiologic and metabolic homeostasis of the host. Our previous studies have demonstrated that oral administration of Lactobacillus frumenti improves epithelial barrier functions and confers diarrhea resistance in early-weaned piglets. However, the metabolic response to L. frumenti administration remains unclear. Then, we conducted simultaneous serum and hepatic metabolomic analyses in early-weaned piglets administered by L. frumenti or phosphate-buffered saline (PBS). Results A total of 100 6-day-old crossbred piglets (Landrace × Yorkshire) were randomly divided into two groups and piglets received PBS (sterile, 2 mL) or L. frumenti (suspension in PBS, 108 CFU/mL, 2 mL) by oral administration once per day from 6 to 20 days of age. Piglets were weaned at 21 days of age. Serum and liver samples for metabolomic analyses were collected at 26 days of age. Principal components analysis (PCA) showed that L. frumenti altered metabolism in serum and liver. Numerous correlations (P < 0.05) were identified among the serum and liver metabolites that were affected by L. frumenti. Concentrations of guanosine monophosphate (GMP), inosine monophosphate (IMP), and uric acid were higher in serum of L. frumenti administration piglets. Pathway analysis indicated that L. frumenti regulated fatty acid and amino acid metabolism in serum and liver. Concentrations of fatty acid β-oxidation related metabolites in serum (such as 3-hydroxybutyrylcarnitine, C4-OH) and liver (such as acetylcarnitine) were increased after L. frumenti administration. Conclusions Our findings suggest that L. frumenti regulates lipid metabolism and amino acid metabolism in the liver of early-weaned piglets, where it promotes fatty acid β-oxidation and energy production. High serum concentrations of nucleotide intermediates, which may be an alternative strategy to reduce the incidence of diarrhea in early-weaned piglets, were further detected. These findings broaden our understanding of the relationships between the gut microbiota and nutrient metabolism in the early-weaned piglets.

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Definition of Essential Amino Acid Residues in the Recognition of a Peptide by a Mouse Monoclonal Antibody

Zeitschrift für Naturforschung C ◽

10.1515/znc-1997-3-423 ◽

1997 ◽

Vol 52 (3-4) ◽

pp. 274-278

Author(s):

Laura Rosanó ◽

Francesca Di Modugno ◽

Giulia Romagnoli ◽

Alberto Chersi

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Critical Role ◽

Binding Activity ◽

Mouse Monoclonal Antibody ◽

Amino Acid Residues ◽

Binding Data ◽

Amino Acid Stretch ◽

Definition Of ◽

Key Residues

AbstractA mouse monoclonal antibody reacting in ELISA with a synthetic peptide representing a linear amino acid stretch of the protein antigen was tested on all overlap ping 5-mer to 9-mer fragments of the peptide, as prepared by multi-pin synthesis. Analysis of the binding data suggests that several residues in the peptide might be relatively unrelevant for recognition, while few others seem to play a critical role as key residues. On the basis of such observations, we attempted to reconstruct an alternative essential epitope by introducing multiple amino acid substitutions in the 9-mer peptide exhibiting the best binding activity, and then tested its ability to be recognized by the monoclonal antibody.

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Heparan Sulfates and Coxsackievirus-Adenovirus Receptor: Each One Mediates Coxsackievirus B3 PD Infection

Journal of Virology ◽

10.1128/jvi.77.18.10071-10077.2003 ◽

2003 ◽

Vol 77 (18) ◽

pp. 10071-10077 ◽

Cited By ~ 55

Author(s):

A. E. Zautner ◽

U. Körner ◽

A. Henke ◽

C. Badorff ◽

M. Schmidtke

Keyword(s):

Amino Acid ◽

Capsid Protein ◽

Critical Role ◽

Chinese Hamster ◽

Coxsackievirus B3 ◽

Productive Infection ◽

Human Hepatocyte Growth Factor ◽

Heparan Sulfates ◽

Capsid Protein Vp1 ◽

Adenovirus Receptor

ABSTRACT Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.

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Amino Acid-Mediated Metabolism: A New Power to Influence Properties of Stem Cells

Stem Cells International ◽

10.1155/2019/6919463 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9

Author(s):

Jilan Liu ◽

Xianyun Qin ◽

Dongfeng Pan ◽

Bin Zhang ◽

Feng Jin

Keyword(s):

Stem Cells ◽

Amino Acid ◽

Cancer Stem Cells ◽

Critical Role ◽

Biological Behavior ◽

Cell Behavior ◽

Self Renewal ◽

Metabolic Characteristics ◽

Therapeutic Modalities ◽

Effect Of Therapy

The self-renewal and differentiation potentials of stem cells are dependent on amino acid (AA) metabolism. We review the literature on the metabolic preference of both cancer and noncancer stem cells. The balance in AA metabolism is responsible for maintaining the functionality of noncancer stem cells, and altering the levels of AAs can influence the malignant biological behavior of cancer stem cells. AAs are considered nutrients participating in metabolism and playing a critical role in maintaining the activity of normal stem cells and the effect of therapy of cancer stem cells. Targeting AA metabolism helps inhibit the stemness of cancer stem cells and remodels the function of normal stem cells. This review summarizes the metabolic characteristics and regulation pathways of AA in different stem cells, not only from the nutritional perspective but also from the genomic perspective that have been reported in the recent five years. In addition, we briefly survey new therapeutic modalities that may help eradicate cancer stem cells by exploiting nutrient deprivation. Understanding AA uptake characteristics helps researchers define the preference for AA in different stem cells and enables clinicians make timely interventions to specifically target the cell behavior.

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