scholarly journals Caspases from Scleractinian Coral Show Unique Regulatory Features

2020 ◽  
Author(s):  
Suman Shrestha ◽  
Jessica Tung ◽  
Robert D. Grinshpon ◽  
Paul Swartz ◽  
Paul T. Hamilton ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Substrate Selection ◽  
Amino Terminal ◽  
Stony Coral ◽  
Initiator Caspases ◽  
Activation Mechanisms ◽  
Immune Challenges ◽  
Caspase 6 ◽  
Biochemical Analyses ◽  
Inflammatory Caspases

AbstractDiseases affecting coral have led to massive decline and altered the community structure of reefs. In response to immune challenges, cnidaria activate apoptotic or autophagic pathways, and the particular pathway correlates with disease sensitivity (apoptosis) or resistance (autophagy). Although cnidaria contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive stony coral, and Porites astreoides, a disease-resistant stony coral. The four caspases are predicted homologs of human caspases-3 and −7, but OfCasp3a and PaCasp7a contain an amino-terminal caspase activation and recruitment domain (CARD) similar to human initiator/inflammatory caspases. In contrast, OfCasp3b and PaCasp3 have short pro-domains, like human effector caspases. We show that OfCasp3a and PaCasp7a are DxxDases, like human caspases-3 and −7, while OfCasp3b and PaCasp3 are more similar to human caspase-6, with VxxDase activity. Our biochemical analyses suggest a mechanism in coral in which the CARD-containing DxxDase is activated on death platforms, but the protease does not directly activate the VxxDase. We also report the first X-ray crystal structure of a coral caspase, that of PaCasp7a determined at 1.57Å resolution. The structure reveals overall conservation of the caspase-hemoglobinase fold in coral as well as an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species, suggesting mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

scholarly journals Caspases from scleractinian coral show unique regulatory features

2020 ◽  
Vol 295 (43) ◽  
pp. 14578-14591
Author(s):  
Suman Shrestha ◽  
Jessica Tung ◽  
Robert D. Grinshpon ◽  
Paul Swartz ◽  
Paul T. Hamilton ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Binding Pocket ◽  
Substrate Selection ◽  
Porites Astreoides ◽  
Orbicella Faveolata ◽  
Initiator Caspases ◽  
Coral Stress ◽  
Biochemical Analyses ◽  
Executioner Caspases

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

scholarly journals Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

2019 ◽  
Vol 476 (22) ◽  
pp. 3475-3492 ◽  
Author(s):  
Robert D. Grinshpon ◽  
Suman Shrestha ◽  
James Titus-McQuillan ◽  
Paul T. Hamilton ◽  
Paul D. Swartz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Pocket ◽  
Gene Duplications ◽  
Substrate Selection ◽  
Charged Amino Acids ◽  
Effector Caspase ◽  
Effector Caspases ◽  
Hydrophobic Binding ◽  
Caspase 6 ◽  
Aspartate Residue

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

scholarly journals Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

2019 ◽  
Author(s):  
Robert D. Grinshpon ◽  
Suman Shrestha ◽  
James Titus-McQuillan ◽  
Paul T. Hamilton ◽  
Paul D. Swartz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Pocket ◽  
Gene Duplications ◽  
Substrate Selection ◽  
Charged Amino Acids ◽  
Effector Caspase ◽  
Effector Caspases ◽  
Hydrophobic Binding ◽  
Caspase 6 ◽  
Aspartate Residue

AbstractApoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases require an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared to caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

Insights into the Structural Aspects of the mGlu Receptor Orthosteric Binding Site

2019 ◽  
Vol 19 (26) ◽  
pp. 2421-2446 ◽  
Author(s):  
Junliang Hao ◽  
Qi Chen
Keyword(s):  
Binding Pocket ◽  
Receptor Subtypes ◽  
X Ray ◽  
Mglu Receptor ◽  
Metabotropic Glutamate ◽  
Amino Terminal ◽  
Subtype Selectivity ◽  
Agonist And Antagonist ◽  
Amino Terminal Domain ◽  
Structural Aspects

The amino terminal domain (ATD) of the metabotropic glutamate (mGlu) receptors contains the orthosteric glutamate recognition site, which is highly conserved across the eight mGlu receptor subtypes. In total, 29 X-ray crystal structures of the mGlu ATD proteins have been reported to date. These structures span across 3 subgroups and 6 subtypes, and include apo, agonist- and antagonist-bound structures. We will discuss the insights gained from the analysis of these structures with the focus on the interactions contributing to the observed group and subtype selectivity for select agonists. Furthermore, we will define the full expanded orthosteric ligand binding pocket (LBP) of the mGlu receptors, and discuss the macroscopic features of the mGlu ATD proteins.

Identification of Critical Residues in the Amino Terminal Domain of the Human NR2B Subunit Involved in the RO 25-6981 Binding Pocket

2003 ◽  
Vol 307 (3) ◽  
pp. 897-905 ◽  
Author(s):  
Pari Malherbe ◽  
Vincent Mutel ◽  
Clemens Broger ◽  
Florent Perin-Dureau ◽  
John A. Kemp ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Nr2b Subunit ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Critical Residues ◽  
Amino Terminal Domain

scholarly journals Receptor-wide Determinants of G Protein Coupling Selectivity in Aminergic GPCRs

2021 ◽  
Author(s):  
Berkay Selçuk ◽  
Ismail Erol ◽  
Serdar Durdağı ◽  
Ogun Adebali
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Md Simulations ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Selective Activation ◽  
G Protein Coupling ◽  
Protein Coupling ◽  
Gpcr Activation ◽  
Activation Mechanisms

AbstractG protein-coupled receptors (GPCRs) induce signal transduction pathways through coupling to four main subtypes of G proteins (Gs, Gi, Gq, G12/13), selectively. However, G protein selective activation mechanisms and residual determinants in GPCRs have remained obscure. Here, we identified conserved G protein selective activation mechanisms determining receptors’ ability to couple to a type of G protein. Herein, we performed an extensive phylogenetic analysis and identified specifically conserved residues for the receptors having similar coupling profiles in each aminergic receptor. By integrating our methodology of differential evolutionary conservation of G protein-specific amino acids with structural analyses, we identified selective activation networks for Gs, Gi1, Go, and Gq. We found that G protein selectivity is determined by not only the G protein interaction site but also other parts of the receptor including the ligand binding pocket. To validate our findings, we further studied an amino acid residue that we revealed as a selectivity-determining in Gs coupling and performed molecular dynamics (MD) simulations. We showed that previously uncharacterized Glycine at position 7×41 plays an important role in both receptor activation and Gs coupling. Finally, we gathered our results into a comprehensive model of G protein selectivity called “sequential switches of activation” describing three main molecular switches controlling GPCR activation: ligand binding, G protein selective activation mechanisms and G protein contact. We believe that our work provides a broader view on receptor-level determinants of G protein coupling selectivity.

Mechanosensitive channels mediate hypoionic shock-induced aminoglycoside potentiation against bacterial persisters by enhancing antibiotic uptake

2021 ◽  
Author(s):  
Boyan Lv ◽  
Youhui Zeng ◽  
Huaidong Zhang ◽  
Zhongyan Li ◽  
Zhaorong Xu ◽  
...  
Keyword(s):  
Cytoplasmic Membrane ◽  
Biological Effects ◽  
Bacterial Pathogens ◽  
Pure Water ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Bacterial Uptake ◽  
Biochemical Analyses

Improving the efficacy of existing antibiotics is a promising strategy for combating antibiotic-resistant/tolerant bacterial pathogens that have become a severe threat to human health. We previously reported that aminoglycoside antibiotics could be dramatically potentiated against stationary-phase Escherichia coli cells under hypoionic shock conditions (i.e., treatment with ion-free solutions), but the underlying molecular mechanism remains unknown. Here we show that mechanosensitive (MS) channels, a ubiquitous protein family sensing mechanical forces of cell membrane, mediate such hypoionic shock-induced aminoglycoside potentiation. Two-minute treatment under conditions of hypoionic shock (e.g., in pure water) greatly enhances the bactericidal effects of aminoglycosides against both spontaneous and triggered E. coli persisters, numerous strains of Gram-negative pathogens in vitro , and Pseudomonas aeruginosa in mice. Such potentiation is achieved by hypoionic shock-enhanced bacterial uptake of aminoglycosides and is linked to hypoionic shock-induced destabilization of the cytoplasmic membrane in E. coli . Genetic and biochemical analyses reveal that MscS-family channels directly and redundantly mediate aminoglycoside uptake upon hypoionic shock and thus potentiation, with MscL channel showing reduced effect. Molecular docking and site-directed mutagenesis analyses reveal a putative streptomycin-binding pocket in MscS, critical for streptomycin uptake and potentiation. These results suggest that hypoionic shock treatment destabilizes the cytoplasmic membrane and thus changes the membrane tension, which immediately activates MS channels that are able to effectively transport aminoglycosides into the cytoplasm for downstream killing. Our findings reveal the biological effects of hypoionic shock on bacteria and can help to develop novel adjuvants for aminoglycoside potentiation to combat bacterial pathogens via activating MS channels.

scholarly journals Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

2016 ◽  
Vol 90 (13) ◽  
pp. 6097-6111 ◽  
Author(s):  
Bethany A. Rhein ◽  
Rachel B. Brouillette ◽  
Grace A. Schaack ◽  
John A. Chiorini ◽  
Wendy Maury
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Ebola Virus ◽  
Virus Entry ◽  
Binding Pocket ◽  
Cell Surface Receptor ◽  
Target Cells ◽  
Virus Binding ◽  
Content Type ◽  
Amino Terminal

ABSTRACTPhosphatidylserine (PtdSer) receptors that are responsible for the clearance of dying cells have recently been found to mediate enveloped virus entry. Ebola virus (EBOV), a member of theFiloviridaefamily of viruses, utilizes PtdSer receptors for entry into target cells. The PtdSer receptors human and murine T-cell immunoglobulin mucin (TIM) domain proteins TIM-1 and TIM-4 mediate filovirus entry by binding to PtdSer on the virion surface via a conserved PtdSer binding pocket within the amino-terminal IgV domain. While the residues within the TIM-1 IgV domain that are important for EBOV entry are characterized, the molecular details of virion–TIM-4 interactions have yet to be investigated. As sequences and structural alignments of the TIM proteins suggest distinct differences in the TIM-1 and TIM-4 IgV domain structures, we sought to characterize TIM-4 IgV domain residues required for EBOV entry. Using vesicular stomatitis virus pseudovirions bearing EBOV glycoprotein (EBOV GP/VSVΔG), we evaluated virus binding and entry into cells expressing TIM-4 molecules mutated within the IgV domain, allowing us to identify residues important for entry. Similar to TIM-1, residues in the PtdSer binding pocket of murine and human TIM-4 (mTIM-4 and hTIM-4) were found to be important for EBOV entry. However, additional TIM-4-specific residues were also found to impact EBOV entry, with a total of 8 mTIM-4 and 14 hTIM-4 IgV domain residues being critical for virion binding and internalization. Together, these findings provide a greater understanding of the interaction of TIM-4 with EBOV virions.IMPORTANCEWith more than 28,000 cases and over 11,000 deaths during the largest and most recent Ebola virus (EBOV) outbreak, there has been increased emphasis on the development of therapeutics against filoviruses. Many therapies under investigation target EBOV cell entry. T-cell immunoglobulin mucin (TIM) domain proteins are cell surface factors important for the entry of many enveloped viruses, including EBOV. TIM family member TIM-4 is expressed on macrophages and dendritic cells, which are early cellular targets during EBOV infection. Here, we performed a mutagenesis screening of the IgV domain of murine and human TIM-4 to identify residues that are critical for EBOV entry. Surprisingly, we identified more human than murine TIM-4 IgV domain residues that are required for EBOV entry. Defining the TIM IgV residues needed for EBOV entry clarifies the virus-receptor interactions and paves the way for the development of novel therapeutics targeting virus binding to this cell surface receptor.

scholarly journals Gasdermin D activity in inflammation and host defense

2019 ◽  
Vol 4 (39) ◽  
pp. eaav1447 ◽  
Author(s):  
Judy Lieberman ◽  
Hao Wu ◽  
Jonathan C. Kagan
Keyword(s):  
Host Defense ◽  
Pore Formation ◽  
Therapeutic Potential ◽  
Interleukin 1 ◽  
Cell Fates ◽  
Secretion Signal ◽  
Amino Terminal ◽  
Distinct Cell ◽  
The Subject ◽  
Inflammatory Caspases

The mechanisms underlying the release of interleukin-1 (IL-1) family cytokines from phagocytes have been the subject of intense investigations for more than 30 years. The absence of an amino-terminal secretion signal from members of this family suggests a previously unknown mechanism of protein secretion that transfers cytosolic IL-1 directly across the plasma membrane into the extracellular space. The pore-forming protein gasdermin D (GSDMD) has emerged as the conduit for IL-1 secretion from the cytosol, serving to induce the release of IL-1 from living (hyperactive) or dead (pyroptotic) cells. In this Review, we discuss the mechanism by which GSDMD pore formation is regulated by the activity of inflammatory caspases, which are commonly associated with inflammasomes. We discuss how GSDMD promotes IL-1 release from hyperactive or pyroptotic cells, with a specific focus on defining how these distinct cell fates associated with GSDMD activity can be regulated. Last, the physiological consequences of GSDMD activity and therapeutic potential of targeting this pore-forming protein are discussed, which highlight the abundance of questions that remain to be answered by the community.

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