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

2020 ◽  
Author(s):  
Gong Qin ◽  
Kim Robinson ◽  
Xu Chenrui ◽  
Zhang Jiawen ◽  
Boo Zhao Zhi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Inner Core ◽  
Inflammatory Diseases ◽  
Structural Features ◽  
Structural Basis ◽  
Card Domain ◽  
Domain Interactions ◽  
Cultured Human Cells ◽  
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 NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular 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 trigger the assembly of distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIINDUPA rings. Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIINDUPA, which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 Å, which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

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 Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region

2017 ◽  
Vol 114 (42) ◽  
pp. 11115-11120 ◽  
Author(s):  
Binnu Gangadharan ◽  
Margaret S. Sunitha ◽  
Souhrid Mukherjee ◽  
Ritu Roy Chowdhury ◽  
Farah Haque ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Troponin T ◽  
Point Mutations ◽  
Structural Features ◽  
Sarcomeric Proteins ◽  
Binding Assays ◽  
Genes Encoding ◽  
The Stability ◽  
Insight Into

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the TNNT2 gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80–180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca2+ in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca2+ sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these TNNT2 mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

scholarly journals Molecular Mechanisms of ZC3H12C/Reg-3 Biological Activity and Its Involvement in Psoriasis Pathology

2021 ◽  
Vol 22 (14) ◽  
pp. 7311
Author(s):  
Mateusz Wawro ◽  
Jakub Kochan ◽  
Weronika Sowinska ◽  
Aleksandra Solecka ◽  
Karolina Wawro ◽  
...  
Keyword(s):  
Family Member ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Association Studies ◽  
Psoriasis Patient ◽  
Genome Wide Association Studies ◽  
Mrna Levels ◽  
Transcript Levels

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1β mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.

scholarly journals Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiuye Li ◽  
W. Michael Babinchak ◽  
Witold K. Surewicz
Keyword(s):  
Amyloid Fibrils ◽  
Low Complexity ◽  
Structural Features ◽  
Protein Fragments ◽  
Hydrophobic Residues ◽  
Backbone Conformation ◽  
Hydrophobic Amino Acids ◽  
Insight Into ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

scholarly journals Current translational potential and underlying molecular mechanisms of necroptosis

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Tamás Molnár ◽  
Anett Mázló ◽  
Vera Tslaf ◽  
Attila Gábor Szöllősi ◽  
Gabriella Emri ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Immune Surveillance ◽  
Kinase Domain ◽  
Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

scholarly journals Antischistosomal Activities of Mefloquine-Related Arylmethanols

2012 ◽  
Vol 56 (6) ◽  
pp. 3207-3215 ◽  
Author(s):  
Katrin Ingram ◽  
William Ellis ◽  
Jennifer Keiser
Keyword(s):  
Body Weight ◽  
Worm Burden ◽  
Structural Features ◽  
Content Type ◽  
Antischistosomal Activity ◽  
Oral Doses ◽  
Related Compounds ◽  
Insight Into

ABSTRACTInteresting antischistosomal properties have been documented for the antimalarial mefloquine, a 4-quinolinemethanol. We evaluated the antischistosomal activities of nine mefloquine-related compounds belonging to the 4-pyridinemethanols, 9-phenanthrenmethanols, and 4-quinolinemethanols. Eight compounds revealed high activities againstSchistosoma mansoni in vitro, with two drugs (the 4-quinolinemethanols WR7573 and WR7930) characterized by significantly lower half-maximal inhibitory concentrations (IC50s) (2.7 and 3.5 μM, respectively) compared to mefloquine (11.4 μM). Mefloquine and WR7930 showed significantly decreased IC50s when incubated in the presence of hemoglobin. High worm burden reductions (WBR) were obtained with enpiroline (WBR, 82.7%; dosage, 200 mg/kg of body weight) and itsthreoisomers (+)-threo(WBR, 100%) and (−)-threo(WBR, 89%) and with WR7930 (WBR, 87%; dosage, 100 mg/kg) against adultS. mansoniin mice. Furthermore, excellentin vitroandin vivoantischistosomal activity was observed for two WR7930-related structures (WR29252 and WR7524). In addition, mefloquine (WBR, 81%), enpiroline (WBR, 77%), and WR7930 (WBR, 100%) showed high activities againstS. haematobiumharbored in mice following single oral doses of 200 mg/kg. These results provide a deeper insight into the structural features of the arylmethanols that rule antischistosomal activity. Further studies should be launched with enpiroline and WR7930.

scholarly journals Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

2019 ◽  
Vol 11 (10) ◽  
pp. 845-859 ◽  
Author(s):  
Alisha N Jones ◽  
Michael Sattler
Keyword(s):  
High Resolution ◽  
Structural Biology ◽  
Recent Progress ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

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