scholarly journals New methods for capturing the mystery lipid, PtdIns5P

2010 ◽  
Vol 428 (3) ◽  
pp. e1-e2 ◽  
Author(s):  
Jonathan M. Backer
Keyword(s):  
Protein Interactions ◽  
Inositol Phosphate ◽  
Coincidence Detection ◽  
Protein Protein Interactions ◽  
Detection Mechanism ◽  
Intact Cells ◽  
New Methods ◽  
Binding Domains ◽  
Biochemical Journal ◽  
Phox Homology

The enormous versatility of phosphatidylinositol as a mediator of intracellular signalling is due to its variable phosphorylation on every combination of the 3′, 4′ and 5′ positions, as well as an even more complex range of phosphorylated products when inositol phosphate is released by phospholipase C activity. The phosphoinositides are produced by distinct enzymes in distinct intracellular membranes, and recruit and regulate downstream signalling proteins containing binding domains [PH (pleckstrin homology), PX (Phox homology), FYVE etc.] that are relatively specific for these lipids. Specific recruitment of downstream proteins presumably involves a coincidence detection mechanism, in which a combination of lipid–protein and protein–protein interactions define specificity. Of the seven intrucellular phosphoinositide, quantification of PtdIns5P levels in intact cells has remained difficult. In this issue of the Biochemical Journal, Sarkes and Rameh describe a novel HPLC-based approach which makes possible an analysis of the subcellular distribution of PtdIns5P and other phosphoinositides.

scholarly journals Single Amino Acid Replacements in RocA Disrupt Protein-Protein Interactions To Alter the Molecular Pathogenesis of Group A Streptococcus

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Paul E. Bernard ◽  
Amey Duarte ◽  
Mikhail Bogdanov ◽  
James M. Musser ◽  
Randall J. Olsen
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Molecular Pathogenesis ◽  
Single Amino Acid ◽  
Accessory Protein ◽  
Protein Protein Interactions ◽  
Tissue Destruction ◽  
Intact Cells ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo. In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.

scholarly journals Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

2006 ◽  
Vol 188 (12) ◽  
pp. 4169-4182 ◽  
Author(s):  
Michael Y. Galperin
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Transcriptional Regulators ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Response Regulators ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Archaeal Species

ABSTRACT CheY-like phosphoacceptor (or receiver [REC]) domain is a common module in a variety of response regulators of the bacterial signal transduction systems. In this work, 4,610 response regulators, encoded in complete genomes of 200 bacterial and archaeal species, were identified and classified by their domain architectures. Previously uncharacterized output domains were analyzed and, in some cases, assigned to known domain families. Transcriptional regulators of the OmpR, NarL, and NtrC families were found to comprise almost 60% of all response regulators; transcriptional regulators with other DNA-binding domains (LytTR, AraC, Spo0A, Fis, YcbB, RpoE, and MerR) account for an additional 6%. The remaining one-third is represented by the stand-alone REC domain (∼14%) and its combinations with a variety of enzymatic (GGDEF, EAL, HD-GYP, CheB, CheC, PP2C, and HisK), RNA-binding (ANTAR and CsrA), protein- or ligand-binding (PAS, GAF, TPR, CAP_ED, and HPt) domains, or newly described domains of unknown function. The diversity of domain architectures and the abundance of alternative domain combinations suggest that fusions between the REC domain and various output domains is a widespread evolutionary mechanism that allows bacterial cells to regulate transcription, enzyme activity, and/or protein-protein interactions in response to environmental challenges. The complete list of response regulators encoded in each of the 200 analyzed genomes is available online at http://www.ncbi.nlm.nih.gov/Complete_Genomes/RRcensus.html .

scholarly journals The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Atsushi Yahashiri ◽  
Matthew A. Jorgenson ◽  
David S. Weiss
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Protein Interactions ◽  
Cell Separation ◽  
Protein Protein Interactions ◽  
Target Proteins ◽  
Bacterial Proteins ◽  
Binding Domains ◽  
Division Site ◽  
Daughter Cell Separation

ABSTRACT Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name “SPOR domain” stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides (“denuded” glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites.

scholarly journals Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide-coupled receptor, but are differentially regulated by guanine nucleotides

1991 ◽  
Vol 280 (1) ◽  
pp. 163-169 ◽  
Author(s):  
M C Sekar ◽  
N Uemura ◽  
D H Coy ◽  
B I Hirschowitz ◽  
K E J Dickinson
Keyword(s):  
Protein Interactions ◽  
Inositol Phosphate ◽  
Membrane Receptors ◽  
Receptor Protein ◽  
Maximum Effect ◽  
Amylase Secretion ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Pancreatic Acini ◽  
Neuromedin B

Bombesin (BB), neuromedin C (NMC) and neuromedin B (NMB) stimulated amylase secretion to similar maximum levels, with EC50 values (concentrations causing 50% of maximum effect) of 0.2, 0.3 and 2 nM respectively. Treatment of pancreatic acini with BB or NMB (10 nM) for 30 min resulted in cross-desensitization of secretory responses to subsequent BB and NMB, but not to acetylcholine, which suggests that NMB and BB activate the same receptor. BB, NMC and NMB stimulated production of similar maximum amounts of inositol mono-, bis- and tris-phosphates, with EC50 values of 3, 5 and 141 nM respectively. The bombesin receptor antagonist [Leu13-psi(CH2NH)Leu14]BB inhibited stimulation of amylase secretion and inositol phosphate formation by BB, NMC and NMB. Binding of 125I-labelled gastrin-releasing peptide (GRP; 200 pM) to rat pancreatic membranes at 22 degrees C was inhibited with relative potencies and IC50 (concn. causing 50% of maximal inhibition; nM) as follows: NMC (0.4) = BB (0.5) greater than NMB (1.8 = GRP (2.6). IC50 values for BB, NMC and NMB inhibition of 125I-GRP binding to intact acini were 5-, 19- and 68-fold higher than their respective values in membranes. The guanine nucleotide analogue guanosine 5′-[beta gamma-imido]triphosphate (Gpp[NH]p) produced rightward shifts of NMC and NMB competition curves by 3.5- and 16-fold respectively, but had little effect on the BB and GRP curves. Elevation of the temperature to 37 degrees C or inclusion of NaCl (40 mM) produced quantitatively similar effects to those of Gpp[NH]p. In the presence of both NaCl and Gpp[NH]p the affinities of peptides for membrane receptors were similar to those for intact cells. Modulation of NMB competition curves by Gpp[NH]p was not attenuated by prior treatment of acini with activated pertussis toxin. These results suggest that BB, NMB and NMC stimulate pancreatic secretion by interaction with a common phosphoinositide-linked receptor. Differences in guanine nucleotide regulation suggest that secretagogue-induced receptor-protein interactions may not be identical for NMB and BB.

scholarly journals Mechanism of Mediator Recruitment by Tandem Gcn4 Activation Domains and Three Gal11 Activator-Binding Domains

2010 ◽  
Vol 30 (10) ◽  
pp. 2376-2390 ◽  
Author(s):  
Eric Herbig ◽  
Linda Warfield ◽  
Lisa Fish ◽  
James Fishburn ◽  
Bruce A. Knutson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Specific Interaction ◽  
Protein Protein Interactions ◽  
Activation Domain ◽  
Activation Domains ◽  
Binding Domains ◽  
Cross Links ◽  
Kix Domain ◽  
The Individual ◽  
Was Gene

ABSTRACT Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.

scholarly journals Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

2020 ◽  
Author(s):  
Christopher R. Carlson ◽  
Jonathan B. Asfaha ◽  
Chloe M. Ghent ◽  
Conor J. Howard ◽  
Nairi Hartooni ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Viral Genome ◽  
Rna Binding ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
N Protein ◽  
Binding Domains ◽  
Genome Transcription ◽  
Liquid Liquid Phase Separation

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell1–3. The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder4. Phosphorylation of the central disordered region is required for normal viral genome transcription5,6, which occurs in a cytoplasmic structure called the replication transcription complex (RTC)7–11. It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates12–15. Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

scholarly journals Selectivity of Protein Interactions along the Aggregation Pathway of α-Synuclein

2021 ◽  
Author(s):  
André D. G. Leitão ◽  
Paulina Rudolffi Soto ◽  
Alexandre Chappard ◽  
Akshay Bhumkar ◽  
Dominic J. B. Hunter ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Self Assembly ◽  
Amyloid Fibrils ◽  
Lewy Bodies ◽  
Coincidence Detection ◽  
Protein Protein Interactions ◽  
Assembly Pathway ◽  
Protein Partners ◽  
Molecular Complexity

AbstractThe aggregation of α-SYN follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson’s Disease in neurons. Whilst α-synuclein is a major contributor to LB, these dense accumulations of protein aggregates and tangles of fibrils contain over 70 different proteins. However, the potential for interactions between these proteins and the different aggregated species of α-SYN is largely unknown. We hypothesized that the proteins present in the Lewy Bodies are trapped or pulled into the aggregates in a hierarchical manner, by binding at specific stages of the aggregation of α-SYN.In this study we uncover a map of interactions of a total of 65 proteins, against different species formed by α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead assay for detection of protein-protein interactions. To access different oligomeric species, we made use of the pathological mutants of α-SYN (A30P, G51D and A53T), which form oligomeric species with distinct properties. Finally, we used bacterially expressed recombinant α-SYN to generate amyloid fibrils and measure interactions with a pool of GFP-tagged potential partners. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are selectively recruited to specific steps in the formation of the LB, explaining their presence in the inclusions. Only a few proteins were found to interact with α-SYN monomers at detectable levels, and only a subset recognizes the oligomeric α-SYN including autophagosomal proteins. We therefore propose a new model for the formation of Lewy Bodies, where selectivity of protein partners at different steps drives the arrangement of these structures, uncovering new ways to modulate aggregation.Significance StatementThe molecular complexity of the Lewy Bodies has been a major hindrance to a bottom-up reconstruction of these inclusions, protein by protein. This work presents an extensive dataset of protein-protein interactions, showing that despite its small size and absence of structure, α-SYN binds to specific partners in the LB, and that there is a clear selectivity of interactions between the different α-SYN species along the self-assembly pathway. We use single-molecule methods to deconvolute number and size of the co-aggregates, to gain detailed information about the mechanisms of interaction. These observations constitute the basis for the elaboration of a global interactome of α-SYN.

scholarly journals Arf•GAPs as Regulators of the Actin Cytoskeleton—An Update

2019 ◽  
Vol 20 (2) ◽  
pp. 442 ◽  
Author(s):  
Christine Tanna ◽  
Louisa Goss ◽  
Calvin Ludwig ◽  
Pei-Wen Chen
Keyword(s):  
Protein Interactions ◽  
Cell Function ◽  
Critical Role ◽  
Focal Adhesions ◽  
Actin Dynamics ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Binding Domains ◽  
Multiple Protein

Arf•GTPase-activating proteins (Arf•GAPs) control the activity of ADP-ribosylation factors (Arfs) by inducing GTP hydrolysis and participate in a diverse array of cellular functions both through mechanisms that are dependent on and independent of their Arf•GAP activity. A number of these functions hinge on the remodeling of actin filaments. Accordingly, some of the effects exerted by Arf•GAPs involve proteins known to engage in regulation of the actin dynamics and architecture, such as Rho family proteins and nonmuscle myosin 2. Circular dorsal ruffles (CDRs), podosomes, invadopodia, lamellipodia, stress fibers and focal adhesions are among the actin-based structures regulated by Arf•GAPs. Arf•GAPs are thus important actors in broad functions like adhesion and motility, as well as the specialized functions of bone resorption, neurite outgrowth, and pathogen internalization by immune cells. Arf•GAPs, with their multiple protein-protein interactions, membrane-binding domains and sites for post-translational modification, are good candidates for linking the changes in actin to the membrane. The findings discussed depict a family of proteins with a critical role in regulating actin dynamics to enable proper cell function.

Frataxin, a molecule of mystery: trading stability for function in its iron-binding site

2010 ◽  
Vol 426 (2) ◽  
pp. e1-e3 ◽  
Author(s):  
Darius J. R. Lane ◽  
Des R. Richardson
Keyword(s):  
Binding Site ◽  
Protein Interactions ◽  
Mitochondrial Protein ◽  
Iron Binding ◽  
Protein Protein Interactions ◽  
Iron Sulfur Cluster ◽  
Downstream Effects ◽  
Biochemical Journal ◽  
And Function ◽  
The Relationship

What are the structural implications for iron binding by frataxin, the mitochondrial protein whose decreased expression results in Friedreich's ataxia? Though frataxin has been shown to be essential for proper handling of iron within mitochondria (e.g. for iron–sulfur cluster and haem biosynthesis), its exact molecular function remains unclear. In this issue of the Biochemical Journal, Correia and colleagues investigate the relationship between structure and function at the putative iron-binding site of Yfh1 (yeast frataxin). Using a host of Yfh1 combination point mutants, the authors observe that the presence of a semi-conserved pocket of negative charge within the ‘acidic ridge’ region (thought to be responsible for iron binding) only mildly enhances Yfh1's ability to bind iron, though it does significantly increase the protein's structural flexibility. The general emerging view is that frataxin's keystone role in mitochondrial iron metabolism depends on iron binding. This appears to have downstream effects on protein–protein interactions that are crucial for frataxin function. The current results reveal a somewhat delicate relationship between iron binding and structural plasticity that may help unravel the enigma of frataxin's metabolic roles.

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