scholarly journals ASC filament formation serves as a signal amplification mechanism for inflammasomes

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Mathias S. Dick ◽  
Lorenzo Sborgi ◽  
Sebastian Rühl ◽  
Sebastian Hiller ◽  
Petr Broz
Keyword(s):  
Signal Amplification ◽  
Cytokine Production ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Inflammasome Activation ◽  
Pyrin Domain ◽  
Efficient Processing ◽  
Amplification Mechanism

Abstract A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASCPYD into filaments and cross-linking of these filaments by ASCCARD. ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASCPYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

scholarly journals Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice

2018 ◽  
Vol 10 (465) ◽  
pp. eaah4066 ◽  
Author(s):  
Richard Gordon ◽  
Eduardo A. Albornoz ◽  
Daniel C. Christie ◽  
Monica R. Langley ◽  
Vinod Kumar ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Nlrp3 Inflammasome ◽  
Lewy Bodies ◽  
Adaptor Protein ◽  
Motor Deficits ◽  
Inflammasome Activation ◽  
Dopaminergic Neurodegeneration ◽  
Pyrin Domain ◽  
Chronic Neuroinflammation ◽  
Dopaminergic Degeneration

Parkinson’s disease (PD) is characterized by a profound loss of dopaminergic neurons in the substantia nigra, accompanied by chronic neuroinflammation, mitochondrial dysfunction, and widespread accumulation of α-synuclein–rich protein aggregates in the form of Lewy bodies. However, the mechanisms linking α-synuclein pathology and dopaminergic neuronal death to chronic microglial neuroinflammation have not been completely elucidated. We show that activation of the microglial NLR family pyrin domain containing 3 (NLRP3) inflammasome is a common pathway triggered by both fibrillar α-synuclein and dopaminergic degeneration in the absence of α-synuclein aggregates. Cleaved caspase-1 and the inflammasome adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) were elevated in the substantia nigra of the brains of patients with PD and in multiple preclinical PD models. NLRP3 activation by fibrillar α-synuclein in mouse microglia resulted in a delayed but robust activation of the NLRP3 inflammasome leading to extracellular interleukin-1β and ASC release in the absence of pyroptosis. Nanomolar doses of a small-molecule NLRP3 inhibitor, MCC950, abolished fibrillar α-synuclein–mediated inflammasome activation in mouse microglial cells and extracellular ASC release. Furthermore, oral administration of MCC950 in multiple rodent PD models inhibited inflammasome activation and effectively mitigated motor deficits, nigrostriatal dopaminergic degeneration, and accumulation of α-synuclein aggregates. These findings suggest that microglial NLRP3 may be a sustained source of neuroinflammation that could drive progressive dopaminergic neuropathology and highlight NLRP3 as a potential target for disease-modifying treatments for PD.

An intersubunit interaction at the active site of D-ribulose-1,5-bisphosphate carboxylase/oxygenase as revealed by cross-linking and site-directed mutagenesis

Biochemistry ◽  
1987 ◽  
Vol 26 (15) ◽  
pp. 4599-4604 ◽  
Author(s):  
Eva H. Lee ◽  
Thomas S. Soper ◽  
Richard J. Mural ◽  
Claude D. Stringer ◽  
Fred C. Hartman
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Bisphosphate Carboxylase

scholarly journals Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy Terminal Extension Essential for Pathogenicity Independent of Pheromone Response

2008 ◽  
Vol 21 (1) ◽  
pp. 110-121 ◽  
Author(s):  
Steven J. Klosterman ◽  
Alfredo D. Martinez-Espinoza ◽  
David L. Andrews ◽  
Jeffrey R. Seay ◽  
Scott E. Gold
Keyword(s):  
Map Kinase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Filamentous Growth ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Sh3 Domains ◽  
Additional Signal ◽  
C Terminus ◽  
Mitogen Activated Protein

Proteins involved in the mitogen-activated protein (MAP) kinase pathway controlling mating, morphogenesis, and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing sterile α motif (SAM) and ras association (RA) domains typical of Ste50-like adaptor proteins found in the fungal phylum Ascomycota, Ubc2 also contains two C-terminal SH3 domains. Yeast two-hybrid assays indicated that Ubc2 interacts with the MAP kinase-kinase kinase Ubc4 via the SAM domains at each of their respective N-termini. Site-directed mutagenesis of ubc2 and complementation analyses revealed that the SAM and RA domains of Ubc2 are essential for filamentous growth. These data support a role for the ascomycete-like N-terminus of Ubc2 in regulating pheromone-responsive mating and morphogenesis analogous to the role of Ste50p in Saccharomyces cerevisiae. In contrast, C-terminal deletion mutants were fully capable of filamentous growth and mating. However, surprisingly, these strains were nonpathogenic. Further, directed mutagenesis of the C-terminus revealed that both SH3 domains are required for pathogenicity. These results suggest that the Basidiomycota have retained the mating and morphogenetic functions of Ste50-type proteins in the N-terminal half of their Ubc2-type adaptors but, additionally, have integrated C-terminal SH3 domains that are critical for additional signal transduction mechanisms, including those that lead to pathogenesis.

scholarly journals Cellular Pyrin Domain-Only Protein 2 Is a Candidate Regulator of Inflammasome Activation

2006 ◽  
Vol 75 (3) ◽  
pp. 1484-1492 ◽  
Author(s):  
Andrea Dorfleutner ◽  
Nicole B. Bryan ◽  
Siera J. Talbott ◽  
Kristin N. Funya ◽  
Stephanie L. Rellick ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Adaptor Protein ◽  
Specific Protein ◽  
Effector Proteins ◽  
Inflammasome Activation ◽  
Pathogen Recognition ◽  
Caspase 1 ◽  
Pyrin Domain ◽  
Specific Effector ◽  
Pathogen Recognition Receptors

ABSTRACT Pyrin domain (PYD) proteins have recently emerged as important signaling molecules involved in the development of innate immunity against intracellular pathogens through activation of inflammatory mediator pathways. ASC is the central adaptor protein, which links pathogen recognition by PYD-containing pathogen recognition receptors, known as PYD-Nod-like receptors (NLR), PAN, PYPAF, NALP, Nod, and Caterpiller proteins, to the activation of downstream effectors, including activation of caspase-1 and NF-κB. Activation of these effectors occurs when specific protein complexes, known as inflammasomes, are formed. PYD signal transduction leads to inflammasome assembly and activation of specific effector proteins. It is modulated by a cellular PYD-only protein (cPOP1), which binds to ASC and interferes with the recruitment of ASC to activated PYD-NLRs. Here we describe the identification and characterization of a second cellular POP (cPOP2), which shows highest homology to the PYD of PAN1. cPOP2 binds to ASC and PAN1, thereby blocking formation of cryopyrin and PAN1-containing inflammasomes, activation of caspase-1, and subsequent processing and secretion of bioactive interleukin-1β. Existence of a second cPOP provides additional insights into inflammasome formation and suggests that POPs might be a common regulatory mechanism to “fine-tune” the activity of specific PYD-NLR family protein-containing inflammasomes.

scholarly journals Rat liver guanidinoacetate methyltransferase. Proximity of cysteine residues at positions 15, 90 and 219 as revealed by site-directed mutagenesis and chemical modification

1991 ◽  
Vol 277 (2) ◽  
pp. 399-406 ◽  
Author(s):  
Y Takata ◽  
T Date ◽  
M Fujioka
Keyword(s):  
Enzyme Activity ◽  
Chemical Modification ◽  
Rat Liver ◽  
Large Change ◽  
Spectroscopic Properties ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Nitrobenzoic Acid

Cys-90 of rat liver guanidinoacetate methyltransferase is a very reactive residue, and chemical modification of this residue results in a large decrease in activity [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. To understand better the role of Cys-90 in catalysis, this residue was replaced with alanine by oligonucleotide-directed mutagenesis. The mutant is active and has kinetic constants similar to those of wild-type, indicating that Cys-90 is not involved in catalysis and substrate binding. The u.v.-absorption, fluorescence and c.d. spectra are also unchanged. Reaction of the mutant with an equimolar amount of 5,5′-dithiobis-(2-nitrobenzoic acid) or 2-nitro-5-thiocyanobenzoic acid results in an almost quantitative disulphide cross-linking between Cys-15 and Cys-21). The same treatment effects disulphide bond formation between Cys-15 and Cys-90 in wild type [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. Since the mutant and wild-type enzymes appear to have similar secondary and tertiary structures, these results suggest that Cys-15, Cys-90 and Cys-219 of the methyltransferase occur spatially close together. The mutant cross-linked between Cys-15 and Cys-219 and the wild-type cross-linked between Cys-15 and Cys-90 show very similar spectroscopic properties. Although treatment of the mutant and wild-type enzymes with equimolar concentrations of 5,5′dithiobis-(2-nitrobenzoic acid) causes a large loss of enzyme activity in each case, kinetic analyses with the modified enzymes suggest that cross-linking of Cys-15 with Cys-90 or Cys-219 does not abolish activity and does not result in a large change in the Michaelis constants. Incubation of the mutant enzyme with excess 2-nitro-5-thiocyanobenzoic acid leads to modification of Cys-207 in addition to Cys-15 and Cys-219. Retention of considerable enzyme activity in the modified enzyme indicates that Cys-207 is also not an essential residue.

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