{alpha}2-Macroglobulin-like protein 1 can conȷugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester [Protein Structure and Folding]
Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have invest...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Seandean Lykke Harwood, Nadia Sukusu Nielsen, Kathrine Teȷlgard Jensen, Peter Kresten Nielsen, Ida B. Thogersen, Jan J. Enghild Tags: Protein Structure and Folding Source Type: research
PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin [Protein Structure and Folding]
In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Rasmus Ree, Laura Kind, Anna Kaziales, Sylvia Varland, Minglu Dai, Klaus Richter, Adrian Drazic, Thomas Arnesen Tags: Enzymology Source Type: research
Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands [Microbiology]
Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the ass...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Laurenne E. Petrie, Allison C. Leonard, Julia Murphy, Georgina Cox Tags: Methods and Resources Source Type: research
Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy [Cell Biology]
In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy ...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Razie Amraei, Tooba Alwani, Rachel Xi-Yeen Ho, Zahra Aryan, Shawn Wang, Nader Rahimi Tags: Cell Biology Source Type: research
Human pancreatic cancer cells under nutrient deprivation are vulnerable to redox system inhibition [Cell Biology]
In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Takefumi Onodera, Isao Momose, Hayamitsu Adachi, Yohko Yamazaki, Ryuichi Sawa, Shun-ichi Ohba, Manabu Kawada Tags: Metabolism Source Type: research
Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes [Microbiology]
We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during Cα-thioether bridge LC–MS/MS fragmentation. Finally, we e...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Clemence Balty, Alain Guillot, Laura Fradale, Clemence Brewee, Benȷamin Lefranc, Christian Herrero, Corine Sandstrom, Jerome Leprince, Olivier Berteau, Alhosna Benȷdia Tags: Enzymology Source Type: research
Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects {beta}-cells from virally mediated lysis [Immunology]
Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cel...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Joshua D. Stafford, Zachary R. Shaheen, Chay Teng Yeo, John A. Corbett Tags: Metabolism Source Type: research
The amphipathic helices of Arfrp1 and Arl14 are sufficient to determine subcellular localizations [Cell Biology]
The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors. By promoting GTP binding, guanine nucleotide exchange factors induce conformational changes of Arf proteins exposing their N-terminal amphipathic helices, which then insert into the membranes to stabilize the membrane association process. Here, we found that the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane–localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging th...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Feng Yang, Tiantian Li, Ziqing Peng, Yang Liu, Yusong Guo Tags: Cell Biology Source Type: research
Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling [Signal Transduction]
The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and R...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Suneet Kaur, Yuqing Chen, Sudha K. Shenoy Tags: Cell Biology Source Type: research
The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+ [Molecular Biophysics]
Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the ...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Shiran Barber-Zucker, Jenny Hall, Afonso Froes, Sofiya Kolusheva, Fraser MacMillan, Raz Zarivach Tags: Protein Structure and Folding Source Type: research
Analysis of {beta}-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism [Enzymology]
We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alte...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Kristina M. J. Aertker, H. T. Henry Chan, Christopher T. Lohans, Christopher J. Schofield Tags: Enzymology Source Type: research
Coupled intra- and interdomain dynamics support domain cross-talk in Pin1 [Signal Transduction]
The functional mechanisms of multidomain proteins often exploit interdomain interactions, or “cross-talk.” An example is human Pin1, an essential mitotic regulator consisting of a Trp–Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. ...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Meiling Zhang, Thomas E. Frederick, Jamie VanPelt, David A. Case, Jeffrey W. Peng Tags: Molecular Biophysics Source Type: research
Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis [Genomics and Proteomics]
This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Francesca Lavatelli, Giulia Mazzini, Stefano Ricagno, Federica Iavarone, Paola Rognoni, Paolo Milani, Mario Nuvolone, Paolo Swuec, Serena Caminito, Masayoshi Tasaki, Antonio Chaves-Sanjuan, Andrea Urbani, Giampaolo Merlini, Giovanni Palladini Tags: Protein Structure and Folding Source Type: research
Functional and structural characterization of allosteric activation of phospholipase Cϵ by Rap1A [Molecular Biophysics]
Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purifie...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Monita Sieng, Arielle F. Selvia, Elisabeth E. Garland-Kuntz, Jesse B. Hopkins, Isaac J. Fisher, Andrea T. Marti, Angeline M. Lyon Tags: Signal Transduction Source Type: research
Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii [Molecular Biophysics]
In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation o...
Source: Journal of Biological Chemistry - December 4, 2020 Category: Chemistry Authors: Tongri Liu, Martine I. Abboud, Rasheduzzaman Chowdhury, Anthony Tumber, Adam P. Hardy, Kerstin Lippl, Christopher T. Lohans, Elisabete Pires, James Wickens, Michael A. McDonough, Christopher M. West, Christopher J. Schofield Tags: Enzymology Source Type: research
