scholarly journals Structural insight into autoinhibition of the NOD-like receptor NLRC4

2014 ◽  
Vol 70 (a1) ◽  
pp. C240-C240
Author(s):  
Zehan Hu ◽  
Jijie Chai
Keyword(s):  
Large Family ◽  
Innate Immune ◽  
Secretion Systems ◽  
Inhibitory Protein ◽  
Danger Signals ◽  
In Vitro Reconstitution ◽  
Structural Insight ◽  
Type 3 ◽  
Insight Into

NOD-like receptor (NLR) proteins constitute a large family of intracellular innate immune receptors. NLRs respond to pathogen-derived signature components or host-derived danger signals, oligomerizing into multiprotein signaling complexes termed inflammasomes that catalyze caspase-1 activation. The NLRC4 (NLR family caspase-recruiting domain (CARD)-containing protein 4) inflammasome is activated by bacterial pathogens carrying the bacterial flagellin or the components of type 3 secretion systems. NAIP (NLR family, apoptosis inhibitory protein) family members dictate the specificity of the NLRC4 inflammasome for different bacterial ligands, with NAIP5/6 and NAIP2 directly recognizing the bacterial flagellin and the T3SS rod protein PrgJ in mice, respectively. We recently solved the crystal structure of NLRC4 in its auotinhibition form. In the current presentation, I discuss the NLRC4 autoinhibtion mechanism and its implications for autoinhbition of other NLR members. I also touch on in vitro reconstitution of the NLRC4 inflammasome and study of its Cryo-EM structure.

scholarly journals Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qin Gong ◽  
Kim Robinson ◽  
Chenrui Xu ◽  
Phuong Thao Huynh ◽  
Kelvin Han Chung Chong ◽  
...  
Keyword(s):  
Cell Death ◽  
Inner Core ◽  
Inflammatory Diseases ◽  
Structural Features ◽  
Structural Basis ◽  
Cultured Human Cells ◽  
Structural Insight ◽  
Sensor Proteins ◽  
Insight Into

AbstractNod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells—a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

scholarly journals Microtubule minus-end stability is dictated by the tubulin off-rate

2019 ◽  
Vol 218 (9) ◽  
pp. 2841-2853 ◽  
Author(s):  
Claire Strothman ◽  
Veronica Farmer ◽  
Göker Arpağ ◽  
Nicole Rodgers ◽  
Marija Podolski ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
In Vitro Reconstitution ◽  
Primary Determinant ◽  
Mean Size ◽  
The Mean ◽  
The Difference ◽  
Insight Into ◽  
Dynamic Organization ◽  
In Cells

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

scholarly journals Production of Staphylococcal Complement Inhibitor (SCIN) and Other Immune Modulators during the Early Stages ofStaphylococcus aureusBiofilm Formation in a Mammalian Cell Culture Medium

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Andi R. Sultan ◽  
Jasper W. Swierstra ◽  
Nicole A. Lemmens-den Toom ◽  
Susan V. Snijders ◽  
Silvie Hansenová Maňásková ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Innate Immune ◽  
Formyl Peptide Receptor ◽  
Complement Inhibitor ◽  
Inhibitory Protein ◽  
Content Type ◽  
Immune Modulators ◽  
Early Stages

ABSTRACTImmune modulators are known to be produced by matured biofilms and during different stages of planktonic growth ofStaphylococcus aureus. Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how doesS. aureusprotect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A. Production was determined duringin vitrobiofilm formation in Iscove's modified Dulbecco's medium at different time points using a competitive Luminex assay and mass spectrometry. Both methods demonstrated the production of the immune modulators SCIN and CHIPS during the early stages of biofilm formation. The green fluorescence protein promoter fusion technology confirmedscn(SCIN) and, to a lesser extent,chp(CHIPS) transcription during the early stages of biofilm formation. Furthermore, we found that SCIN could inhibit human complement activation induced by early biofilms, indicating thatS. aureusis able to modulate the innate immune system already during the early stages of biofilm formationin vitro. These results form a stepping stone toward elucidating the role of immune modulators in the establishment of biofilmsin vivoand present opportunities to develop preventive strategies.

scholarly journals In Vivo Discrimination of Type 3 Secretion System-Positive and -Negative Pseudomonas aeruginosa via a Caspase-1-Dependent Pathway

2010 ◽  
Vol 78 (11) ◽  
pp. 4744-4753 ◽  
Author(s):  
Tamding Wangdi ◽  
Lilia A. Mijares ◽  
Barbara I. Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Immune System ◽  
Innate Immune System ◽  
Interleukin 1 ◽  
Innate Immune ◽  
Secretion Systems ◽  
Caspase 1 ◽  
Molecular Patterns ◽  
Type 3

ABSTRACT Microbe-associated molecular patterns are recognized by Toll-like receptors of the innate immune system. This recognition enables a rapid response to potential pathogens but does not clearly provide a way for the innate immune system to discriminate between virulent and avirulent microbes. We find that pulmonary infection of mice with type 3 translocation-competent Pseudomonas aeruginosa triggers a rapid inflammatory response, while infection with isogenic translocation-deficient mutants does not. Discrimination between translocon-positive and -negative bacteria requires caspase-1 activity in bone marrow-derived cells and interleukin-1 receptor signaling. Thus, the activation of caspase-1 by bacteria expressing type 3 secretion systems allows for rapid recognition of bacteria expressing conserved functions associated with virulence.

scholarly journals A Primed Subpopulation of Bacteria Enables Rapid Expression of the Type 3 Secretion System in Pseudomonas aeruginosa

mBio ◽  
2021 ◽  
Author(s):  
Christina K. Lin ◽  
Daniel S. W. Lee ◽  
Saria McKeithen-Mead ◽  
Thierry Emonet ◽  
Barbara Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Secretion System ◽  
Innate Immune ◽  
Secretion Systems ◽  
Content Type ◽  
Acute Infections ◽  
A Cell ◽  
Type 3 Secretion System ◽  
Type 3 ◽  
Specific Virulence

The expression of specific virulence traits is strongly associated with Pseudomonas aeruginosa ’s success in establishing acute infections but is thought to carry a cost for bacteria. Producing multiprotein secretion systems or motility organelles is metabolically expensive and can target a cell for recognition by innate immune system receptors that recognize structural components of the type 3 secretion system (T3SS) or flagellum.

scholarly journals Aquifex aeolicus PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase

2002 ◽  
Vol 184 (23) ◽  
pp. 6465-6471 ◽  
Author(s):  
Timothy J. Herdendorf ◽  
Darrell R. McCaslin ◽  
Katrina T. Forest
Keyword(s):  
Specific Activity ◽  
Protein A ◽  
Large Family ◽  
Size Exclusion ◽  
Sedimentation Equilibrium ◽  
Binding Motif ◽  
Aquifex Aeolicus ◽  
Secretion Systems ◽  
Surface Motility

ABSTRACT Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a ≈5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed.

scholarly journals Novel Structural Insight into Inhibitors of Heme Oxygenase-1 (HO-1) by New Imidazole-Based Compounds: Biochemical and In Vitro Anticancer Activity Evaluation

Molecules ◽  
2018 ◽  
Vol 23 (5) ◽  
pp. 1209 ◽  
Author(s):  
Khaled Greish ◽  
Loredana Salerno ◽  
Reem Al Zahrani ◽  
Emanuele Amata ◽  
Maria Modica ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Heme Oxygenase ◽  
Heme Oxygenase 1 ◽  
Activity Evaluation ◽  
Structural Insight ◽  
Insight Into

scholarly journals Bacteriophage-Resistant Salmonella rissen: An In Vitro Mitigated Inflammatory Response

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2468
Author(s):  
Rosanna Capparelli ◽  
Paola Cuomo ◽  
Marina Papaianni ◽  
Cristina Pagano ◽  
Angela Michela Immacolata Montone ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Innate Immune ◽  
Health Concern ◽  
Phage Resistance ◽  
Potential Cost ◽  
Structural Modifications ◽  
Selection For ◽  
Secretory System ◽  
Type 3

Non-typhoid Salmonella (NTS) represents one of the major causes of foodborne diseases, which are made worse by the increasing emergence of antibiotic resistance. Thus, NTS are a significant and common public health concern. The purpose of this study is to investigate whether selection for phage-resistance alters bacterial phenotype, making this approach suitable for candidate vaccine preparation. We therefore compared two strains of Salmonella enterica serovar Rissen: RR (the phage-resistant strain) and RW (the phage-sensitive strain) in order to investigate a potential cost associated with the bacterium virulence. We tested the ability of both RR and RW to infect phagocytic and non-phagocytic cell lines, the activity of virulence factors associated with the main Type-3 secretory system (T3SS), as well as the canonic inflammatory mediators. The mutant RR strain—compared to the wildtype RW strain—induced in the host a weaker innate immune response. We suggest that the mitigated inflammatory response very likely is due to structural modifications of the lipopolysaccharide (LPS). Our results indicate that phage-resistance might be exploited as a means for the development of LPS-based antibacterial vaccines.

scholarly journals Modeling a disease-correlated tubulin mutation in budding yeast reveals insight into MAP-mediated dynein function

2021 ◽  
pp. mbc.E21-05-0237
Author(s):  
E. Denarier ◽  
K.H. Ecklund ◽  
G. Berthier ◽  
A. Favier ◽  
E.T. O'Toole ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Budding Yeast ◽  
Neurological Diseases ◽  
Large Family ◽  
Disease Etiology ◽  
Tubulin Isotype ◽  
Insight Into ◽  
Microtubule Function

Mutations in the genes that encode α- and β-tubulin underlie many neurological diseases, most notably malformations in cortical development (MCD). In addition to revealing the molecular basis for disease etiology, studying such mutations can provide insight into microtubule function, and the role of the large family of microtubule effectors. In this study, we use budding yeast to model one such mutation – Gly436Arg in α-tubulin, which is causative of MCD – in order to understand how it impacts microtubule function in a simple eukaryotic system. Using a combination of in vitro and in vivo methodologies, including live cell imaging and electron tomography, we find that the mutant tubulin incorporates into microtubules, causes a shift in α-tubulin isotype usage, and dramatically enhances dynein activity, which leads to spindle positioning defects. We find that the basis for this latter phenotype is an impaired interaction between She1 – a dynein inhibitor – and the mutant microtubules. In addition to revealing the natural balance of α-tubulin isotype utilization in cells, our results provide evidence of an impaired interaction between microtubules and a dynein regulator as a consequence of a tubulin mutation, and sheds light on a mechanism that may be causative of neurodevelopmental diseases. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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