Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

Vaidya, Sravanti; Velázquez-Delgado, Elih M.; Abbruzzese, Genevieve; Hardy, James L.

doi:10.1016/j.jmb.2010.11.031

Cited by 52 publications

(103 citation statements)

References 66 publications

(65 reference statements)

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“…However, in cathepsin B (35), the prodomain is only weakly associated with the enzyme, through five hydrogen bonds, and it crosses the active site in the opposite orientation to that observed in MCA2. Structural elucidation of a caspase prodomain is yet to be achieved, but it has been reported to be flexible, and hence disordered, in the crystal structures (36). In contrast, the N-terminal domain of MCA2 was both visible and highly ordered in our crystal structures, with its position clearly showing that it would be required to undergo a conformational shift to allow substrates access into the active site.…”

Section: Resultsmentioning

confidence: 73%

Crystal structure of a Trypanosoma brucei metacaspase

McLuskey

Rudolf

Proto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

110

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Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded β-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.apoptosis | clan CD | parasite | X-ray crystallography P rogrammed cell death (PCD) is essential for animal development and the maintenance of adult tissues. PCD itself is a regulated process, and in animals, apoptosis is controlled through the action of caspases, aspartic acid-specific cysteine peptidases (1). PCD is also essential for plant development (2), and multiple markers for apoptosis have been described in yeast and a broad range of protozoan parasites (3), yet these organisms do not encode caspases in their genomes; thus, alternative pathways must have evolved for caspase-independent cell death to be regulated. In recent years, attention has focused on the metacaspases, which are a highly conserved group of caspase-like cysteine peptidases found in plants, fungi, and protozoa but not in the metazoa (4). In plants, metacaspases are essential for embryogenesis (5, 6) and have been shown to act in antagonistic relationships, functioning as both positive and negative regulators of PCD (7). In yeast and some protozoa, metacaspases have been implicated as mediators of cell death in response to oxidative stress and environmental change (3,(8)(9)(10).Various attempts have been made to draw parallels between caspases and metacaspases in respect to structure, activation, and function (11). However, two striking differences between metacaspases and caspases are their substrate specificities and requirements for activation. Metacaspases have arginine/lysine specificity, do not necessarily require proces...

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Section: Resultsmentioning

confidence: 73%

Crystal structure of a Trypanosoma brucei metacaspase

McLuskey

Rudolf

Proto

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

110

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“…Generation of Caspase-6 Variants-The wild-type caspase-6 constructs consisted of a synthetic gene encoding the Escherichia coli codon-optimized, full-length, C-terminal His 6 -tagged human CASPASE-6 gene comprising amino acids 1-293 plus His 6 ligated into the NdeI/BamHI sites of the pET11a vector (Stratagene) or from a constitutive two-chain (CT) 3 version of the synthetic CASPASE-6 gene (Celtek Bioscience) (16). Amino acid substitutions were introduced by the QuikChange site-directed mutagenesis method (Stratagene) in the fulllength construct or the CT construct.…”

Section: Methodsmentioning

confidence: 99%

“…3B). Caspase-6 is the only caspase known to exist in a latent, highly helical conformation where the 60s and 130s helices are elongated and the 90s helix is rotated out relative to the canonical fold (16,18). We soaked zinc into crystals of this helical conformation and found that the zinc-bound structure remains in the latent helical form (Fig.…”

Section: Zinc Is the Only Transition Metal To Inhibit Caspase-6-amentioning

confidence: 99%

“…Cleavage of the intersubunit linker produces two nascent loops that participate in a four-loop substrate-binding groove bundle. In caspase-6 this region is capable of a strand-to-helix transition that results in a unique helical conformation (16), which cannot be attained by any other caspase (17,18). Caspase-6 is capable of autoactivation by self-cleavage of the intersubunit linker at position 193 following the sequence TEVD (19).…”

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confidence: 99%

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Zinc-mediated Allosteric Inhibition of Caspase-6

Velázquez-Delgado

Hardy

2012

Journal of Biological Chemistry

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Background: Caspase-6 is a critical factor in neurodegeneration, which is regulated by zinc binding. Results: Caspase-6 is inhibited by zinc and binds one zinc/monomer at an exosite distal from the active site. Conclusion: Zinc allosterically inhibits caspase-6 by locking it into a naturally occurring, inactive, and extended helical conformation. Significance: The allosteric inhibition observed in the presence of zinc may aid in the development of allosteric caspase-6 drugs.

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“…To further determine how palmitoylation of CASP6 might affect its processing and activation (Figure 4a), we utilized computational molecular modelling based on the published crystal structures of CASP6. 24,25,[49][50][51][52][53][54][55] Generation of the model of CASP6 required the construction of coordinates of loop 2 (L2), which forms the substrate-binding groove (active site) and is not resolved in any of the several crystal structures available, presumably owing to high flexibility. The structure of CASP6 has a six-stranded β-sheet flanked by five α-helices and two small β-strands.…”

Section: Resultsmentioning

confidence: 99%

Palmitoylation of caspase-6 by HIP14 regulates its activation

et al. 2016

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Caspase-6 (CASP6) has an important role in axonal degeneration during neuronal apoptosis and in the neurodegenerative diseases Alzheimer and Huntington disease. Decreasing CASP6 activity may help to restore neuronal function in these and other diseases such as stroke and ischemia, where increased CASP6 activity has been implicated. The key to finding approaches to decrease CASP6 activity is a deeper understanding of the mechanisms regulating CASP6 activation. We show that CASP6 is posttranslationally palmitoylated by the palmitoyl acyltransferase HIP14 and that the palmitoylation of CASP6 inhibits its activation. Palmitoylation of CASP6 is decreased both in Hip14 −/− mice, where HIP14 is absent, and in YAC128 mice, a model of Huntington disease, where HIP14 is dysfunctional and where CASP6 activity is increased. Molecular modeling suggests that palmitoylation of CASP6 may inhibit its activation via steric blockage of the substrate-binding groove and inhibition of CASP6 dimerization, both essential for CASP6 function. Our studies identify palmitoylation as a novel CASP6 modification and as a key regulator of CASP6 activity.

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Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

Cited by 52 publications

References 66 publications

Crystal structure of a Trypanosoma brucei metacaspase

Crystal structure of a Trypanosoma brucei metacaspase

Zinc-mediated Allosteric Inhibition of Caspase-6

Palmitoylation of caspase-6 by HIP14 regulates its activation

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