scholarly journals The Rice Plastidial Phosphorylase Participates Directly in Both Sink and Source Processes

2020 ◽  
Author(s):  
Kaan Koper ◽  
Seon-Kap Hwang ◽  
Magnus Wood ◽  
Salvinder Singh ◽  
Asaph Cousins ◽  
...  
Keyword(s):  
Co2 Assimilation ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Starch Phosphorylase ◽  
Higher Plant ◽  
Dual Roles ◽  
Terminal Electron Acceptor ◽  
Spectrometry Analysis ◽  
Rice Mutant ◽  
Wild Type Phenotype

Abstract The plastidial starch phosphorylase (Pho1) functions in starch metabolism. A distinctive structural feature of the higher Pho1 is a 50–82-amino-acid long peptide (L50–L82), which is absent in phosphorylases from non-plant organisms. To study the function of the rice Pho1 L80 peptide, we complemented a pho1− rice mutant (BMF136) with the wild-type Pho1 gene or with a Pho1 gene lacking the L80 region (Pho1ΔL80). While expression of Pho1 in BMF136 restored normal wild-type phenotype, the introduction of Pho1ΔL80 enhanced the growth rate and plant productivity above wild-type levels. Mass spectrometry analysis of proteins captured by anti-Pho1 showed the surprising presence of PsaC, the terminal electron acceptor/donor subunit of photosystem I (PSI). This unexpected interaction was substantiated by reciprocal immobilized protein pull-down assays of seedling extracts and supported by the presence of Pho1 on isolated PSI complexes resolved by blue-native gels. Spectrophotometric studies showed that Pho1ΔL80 plants exhibited modified PSI and enhanced CO2 assimilation properties. Collectively, these findings indicate that the higher plant Pho1 has dual roles as a potential modulator of source and sink processes.

scholarly journals The Rice Plastidial Phosphorylase Participates Directly In Both Sink And Source Processes

2020 ◽  
Author(s):  
Kaan Koper ◽  
Seon-Kap Hwang ◽  
Magnus Wood ◽  
Salvinder Singh ◽  
Asaph Cousins ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Starch Phosphorylase ◽  
Higher Plant ◽  
Dual Roles ◽  
Terminal Electron Acceptor ◽  
Spectrometry Analysis ◽  
Rice Mutant ◽  
Blue Native ◽  
Source And Sink

AbstractA distinctive structural feature of the higher plant plastidial starch phosphorylase (Pho1) is a 50 to 82 amino acid long peptide (L50 - L82), which is absent in phosphorylases from non-plant organisms. To study the function of the rice Pho1 L80 peptide, we complemented a pho1− rice mutant (BMF136) with the wildtype Pho1 gene or with a Pho1 gene lacking the L80 region (Pho1ΔL80). While expression of Pho1 in BMF136 restored normal wildtype phenotype, the introduction of Pho1ΔL80 enhanced growth rate and plant productivity above wildtype levels. Mass spectrometry analysis of proteins captured by anti-Pho1 showed the surprising presence of PsaC, the terminal electron acceptor/donor subunit of photosystem I (PSI). This unexpected interaction was substantiated by reciprocal immobilized protein pulldown assays of seedling extracts and supported by the presence of Pho1 on isolated PSI complexes resolved by blue native gels. Spectrophotometric studies showed that Pho1ΔL80 plants exhibited modified PSI and enhanced CO2 assimilation properties. Collectively, these findings indicate that the higher plant Pho1 has dual roles as a potential modulator of source and sink processes.

scholarly journals Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer

2021 ◽  
Vol 118 (21) ◽  
pp. e2016904118
Author(s):  
Derek K. Cheng ◽  
Tobiloba E. Oni ◽  
Jennifer S. Thalappillil ◽  
Youngkyu Park ◽  
Hsiu-Chi Ting ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Clinical Success ◽  
Treatment Options ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Ras Signaling ◽  
Wild Type ◽  
Feedback Mechanisms ◽  
Spectrometry Analysis ◽  
Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

scholarly journals Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

2010 ◽  
Vol 192 (8) ◽  
pp. 2044-2052 ◽  
Author(s):  
Jyl S. Matson ◽  
Hyun Ju Yoo ◽  
Kristina Hakansson ◽  
Victor J. DiRita
Keyword(s):  
Antimicrobial Peptides ◽  
Vibrio Cholerae ◽  
Lipid A ◽  
Polymyxin B ◽  
Mass Spectrometry Analysis ◽  
Peptide Antibiotic ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Antimicrobial Peptide Resistance ◽  
El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

scholarly journals Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

2010 ◽  
Vol 192 (18) ◽  
pp. 4651-4659 ◽  
Author(s):  
Wendy D. Smith ◽  
Jonathan A. Pointon ◽  
Emily Abbot ◽  
Hae Joo Kang ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Human Keratinocytes ◽  
Mass Spectrometry Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Protein Adhesion ◽  
Culture Supernatants

ABSTRACT Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

scholarly journals Modification of Lipooligosaccharide with Phosphoethanolamine by LptA in Neisseria meningitidis Enhances Meningococcal Adhesion to Human Endothelial and Epithelial Cells

2008 ◽  
Vol 76 (12) ◽  
pp. 5777-5789 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Russel W. Carlson ◽  
Artur Muszynski ◽  
Biswa Choudhury ◽  
Kwang Sik Kim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Meningitidis ◽  
Lipid A ◽  
Inner Core ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Epithelial Cell Lines

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

scholarly journals Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Valentina M. T. Mayer ◽  
Markus B. Tomek ◽  
Rudolf Figl ◽  
Marina Borisova ◽  
Isabel Hottmann ◽  
...  
Keyword(s):  
Natural Habitat ◽  
Indirect Evidence ◽  
Mass Spectrometry Analysis ◽  
Membrane Transporter ◽  
Wild Type ◽  
Tannerella Forsythia ◽  
Inner Membrane ◽  
Oral Pathogen ◽  
Spectrometry Analysis ◽  
E Coli

Abstract Background The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(−peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. Results We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(−peptides) and GlcNAc-MurNAc(−peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. Conclusion T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium’s inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

scholarly journals Physiological Adaptation of Desulfitobacterium hafniense Strain TCE1 to Tetrachloroethene Respiration

2011 ◽  
Vol 77 (11) ◽  
pp. 3853-3859 ◽  
Author(s):  
Laure Prat ◽  
Julien Maillard ◽  
Régis Grimaud ◽  
Christof Holliger
Keyword(s):  
Electron Acceptor ◽  
Growth Conditions ◽  
Mass Spectrometry Analysis ◽  
Physiological Adaptation ◽  
Terminal Electron Acceptor ◽  
Organohalide Respiration ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Metabolic Versatility ◽  
Desulfitobacterium Hafniense

ABSTRACTDesulfitobacteriumspp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells ofDesulfitobacterium hafniensestrain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest thatD. hafniensestrain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.

scholarly journals PPP2R5D Promotes Hepatitis C Virus Infection Through Binding to NS5B Protein

2021 ◽  
Author(s):  
Muhammad Ikram Anwar ◽  
Ni Li ◽  
Qing Zhou ◽  
Mingxiao Chen ◽  
Chengguang Hu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Hcv Infection ◽  
Mass Spectrometry Analysis ◽  
Enhancement Effect ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Adaptive Mutations

Abstract Background: Hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis C, end-stage liver diseases, and hepatocellular carcinoma. The development of infectious HCV cell culture systems primarily relied on the replication enhancement effect of adaptive mutations. Although the mode of action may vary, those adaptive mutations could direct the study of virus-host interactions required for efficient virus infection. We previously identified a substitution D559G in NS5B (RNA dependent RNA polymerase) critical for the replication of HCV genomes. In this study, we set out to study whether D559G-NS5B specifically interacted with some host factors crucial for HCV infection.Methods: Through mass spectrometry analysis of immunoprecipitation mixture of ectopically expressed wild-type and D559G-mutated NS5B, we identified candidate factors showing potential interactions with NS5B and D559G-NS5B. The requirement of selected host factor in HCV infection in vitro was demonstrated by gene knockout, overexpression, virus infection, and co-immunoprecipitation approaches.Results: From the results of immunoprecipitation and mass spectrometry analysis, we selected protein phosphatase 2 regulatory subunit B’delta (PPP2R5D) for further characterization. Co-immunoprecipitation confirmed that both wild-type and D559G NS5B proteins interacted with PPP2R5D, but the interaction between D559G-NS5B and PPP2R5D was more efficient. Silencing of PPP2R5D decreased HCV infection, and knockout of PPP2R5D nearly eliminated HCV infection in Huh7.5 cells. Transient and stably overexpression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that seen for wild-type Huh7.5 cells. Conclusions: PPP2R5D is required for HCV infection in cultured hepatoma cells, and PPP2R5D may function through binding to HCV NS5B. The underlying mechanism of PPP2R5D in the complete HCV life cycle requires further investigation.

scholarly journals Deficiency of ITGAM Attenuates Experimental Abdominal Aortic Aneurysm in Mice

2021 ◽  
Author(s):  
Min Zhou ◽  
Xia Wang ◽  
Yiqin Shi ◽  
Yong Ding ◽  
Xu Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Abdominal Aortic Aneurysm ◽  
Aortic Aneurysm ◽  
Transendothelial Migration ◽  
Mass Spectrometry Analysis ◽  
Integrin Subunit ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Abdominal Aortic

Background Integrin αM (CD11b), which is encoded by the Integrin Subunit Alpha M (ITGAM) gene, is not only a surface marker of monocytes but also an essential adhesion molecule. In this study, we investigated the effect of CD11b on experimental abdominal aortic aneurysm and the potential underlying mechanisms. Methods and Results The incidence of abdominal aortic aneurysm was not significantly lower in ITGAM(‐/‐) mice than in control mice. Nevertheless, knockout of CD11b reduced the maximum abdominal aortic diameter, macrophage infiltration, matrix metalloproteinase‐9 expression, and elastin and collagen degradation. Additionally, lower expression of IL‐6 was found in both the peripheral blood and abdominal aortas of ITGAM(‐/‐) mice, indicating a biological correlation between CD11b and the inflammatory response in abdominal aortic aneurysm. In vitro, the number of ITGAM(‐/‐) bone marrow–derived macrophages (BMDMs) that adhered to endothelial cells was significantly lower than the number of wild‐type BMDMs. Moreover, the CD11b monoclonal antibody and CD11b agonist leukadherin‐1 decreased and increased the number of adherent wild‐type BMDMs, respectively. Through RNA sequencing, genes associated with leukocyte transendothelial migration were found to be downregulated in ITGAM(‐/‐) BMDMs. Furthermore, immunoprecipitation–mass spectrometry analysis predicted that the Akt pathway might be responsible for the impaired transmigratory ability of ITGAM(‐/‐) BMDMs. The reduced activation of Akt was then confirmed, and the Akt agonist SC79 partially rescued the transendothelial migratory function of ITGAM(‐/‐) BMDMs. Conclusions CD11b might promote the development and progression of abdominal aortic aneurysm by mediating the endothelial cells adhesion and transendothelial migration of circulating monocytes/macrophages.

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