Sequential Unfolding Mechanisms of Monomeric Caspases.

Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood.

Conserved folding landscape of monomeric initiator caspases.

The apoptotic caspase subfamily evolved into two subfamilies-monomeric initiators and dimeric effectors; both subfamilies share a conserved caspase-hemoglobinase fold with a protease domain containing a large subunit and a small subunit. Sequence variations in the conserved caspase-hemoglobinase fold resulted in changes in oligomerization, enzyme specificity, and regulation, making caspases an excellent model for examining the mechanisms of molecular evolution in fine-tuning structure, function, and allosteric regulation. We examined the urea-induced equilibrium folding/unfolding of two initiator caspases, monomeric caspase-8 and cFLIP, over a broad pH range.

Sequential unfolding mechanisms of monomeric caspases.

Caspases are evolutionarily conserved cysteinyl proteases that are integral in cell development and apoptosis. All apoptotic caspases evolved from a common ancestor into two distinct subfamilies with either monomeric (initiators) or dimeric (effectors) oligomeric states. The regulation of apoptosis is influenced by the activation mechanism of the two subfamilies, but the evolution of the well-conserved caspase-hemoglobinase fold into the two subfamilies is not well understood.

Comparing the folding landscapes of evolutionarily divergent procaspase-3.

All caspases evolved from a common ancestor and subsequently developed into two general classes, inflammatory or apoptotic caspases. The caspase-hemoglobinase fold has been conserved throughout nearly one billion years of evolution and is utilized for both the monomeric and dimeric subfamilies of apoptotic caspases, called initiator and effector caspases, respectively. We compared the folding and assembly of procaspase-3b from zebrafish to that of human effector procaspases in order to examine the conservation of the folding landscape.

Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold.

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1β (IL-1β). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1β.

Evolution of the folding landscape of effector caspases.

Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases [PCPs]) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor, whereas others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor (CA) into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases).

Remodeling hydrogen bond interactions results in relaxed specificity of Caspase-3.

Caspase (or cysteinyl-aspartate specific proteases) enzymes play important roles in apoptosis and inflammation, and the non-identical but overlapping specificity profiles (that is, cleavage recognition sequence) direct cells to different fates. Although all caspases prefer aspartate at the P1 position of the substrate, the caspase-6 subfamily shows preference for valine at the P4 position, while caspase-3 shows preference for aspartate. In comparison with human caspases, caspase-3a from zebrafish has relaxed specificity and demonstrates equal selection for either valine or aspartate at the P4 position.

Caspases from scleractinian coral show unique regulatory features.

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.

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

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.

The CaspBase: a curated database for evolutionary biochemical studies of caspase functional divergence and ancestral sequence inference.

Sequence databases are powerful tools for the contemporary scientists' toolkit. However, most functional annotations in public databases are determined computationally and are not verified by a human expert. While hypotheses generated from computational studies are now amenable to experimentation, the quality of the results relies on the quality of input data.

Modifications to a common phosphorylation network provide individualized control in caspases.

Caspase-3 activation and function have been well-defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation. Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK (mitogen-activated protein kinase) promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes.

Phage display and structural studies reveal plasticity in substrate specificity of caspase-3a from zebrafish.

The regulation of caspase-3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases.

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.

Caspase Allostery and Conformational Selection.

The role of caspase proteases in regulated processes such as apoptosis and inflammation has been studied for more than two decades, and the activation cascades are known in detail. Apoptotic caspases also are utilized in critical developmental processes, although it is not known how cells maintain the exquisite control over caspase activity in order to retain subthreshold levels required for a particular adaptive response while preventing entry into apoptosis. In addition to active site-directed inhibitors, caspase activity is modulated by post-translational modifications or metal binding to allosteric sites on the enzyme, which stabilize inactive states in the conformational ensemble.

Modifying caspase-3 activity by altering allosteric networks.

Caspases have several allosteric sites that bind small molecules or peptides. Allosteric regulators are known to affect caspase enzyme activity, in general, by facilitating large conformational changes that convert the active enzyme to a zymogen-like form in which the substrate-binding pocket is disordered. Mutations in presumed allosteric networks also decrease activity, although large structural changes are not observed.

Redesigning the procaspase-8 dimer interface for improved dimerization.

Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain.

Lengthening the intersubunit linker of procaspase 3 leads to constitutive activation.

The conformational ensemble of procaspase 3, the primary executioner in apoptosis, contains two major forms, inactive and active, with the inactive state favored in the native ensemble. A region of the protein known as the intersubunit linker (IL) is cleaved during maturation, resulting in movement of the IL out of the dimer interface and subsequent active site formation (activation-by-cleavage mechanism). We examined two models for the role of the IL in maintaining the inactive conformer, an IL-extension model versus a hydrophobic cluster model, and we show that increasing the length of the IL by introducing 3-5 alanines results in constitutively active procaspases.

Slow folding and assembly of a procaspase-3 interface variant.

Caspases execute apoptosis and exist in the cell as inactive zymogens (procaspases) prior to activation. Initiator procaspases are monomers that must dimerize for activation, while effector procaspases, such as procaspase-3, are stable dimers that must be processed for activation. The dimer interface regions of the two subfamilies are different, although the role of the interface in oligomerization is not known.

Death by caspase dimerization.

Controlled cell death, or apoptosis, occurs in response to many different environmental stimuli. The apoptotic cascade that occurs within the cell in response to these cues leads to morphological and biochemical changes that trigger the dismantling and packaging of the cell. Caspases are a family of cysteine-dependent aspartate-directed proteases that play an integral role in the cascade that leads to apoptosis.

Allosteric modulation of caspase 3 through mutagenesis.

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants.

A bifunctional allosteric site in the dimer interface of procaspase-3.

The dimer interface of caspase-3 contains a bifunctional allosteric site in which the enzyme can be activated or inactivated, depending on the context of the protein. In the mature caspase-3, the binding of allosteric inhibitors to the interface results in an order-to-disorder transition in the active site loops. In procaspase-3, by contrast, the binding of allosteric activators to the interface results in a disorder-to-order transition in the active site.

Phylogeny and adaptive evolution of the brain-development gene microcephalin (MCPH1) in cetaceans.

Background: Representatives of Cetacea have the greatest absolute brain size among animals, and the largest relative brain size aside from humans. Despite this, genes implicated in the evolution of large brain size in primates have yet to be surveyed in cetaceans.

Results: We sequenced ~1240 basepairs of the brain development gene microcephalin (MCPH1) in 38 cetacean species.

Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3.

Interactions between loops 2, 2' and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall conformational stability and in maintaining the active site environment.

The potential for caspases in drug discovery.

Caspases are a family of proteases that are involved in the execution of apoptosis and the inflammatory response. A plethora of diseases occur as a result of the dysregulation of apoptosis and inflammation, and caspases have been targeted as a therapeutic strategy to halt the progression of such diseases. Hundreds of peptide and peptidomimetic inhibitors have been designed and tested, but only a few have advanced to clinical trials because of poor drug-like properties and pharmacological constraints.

Folding and assembly kinetics of procaspase-3.

Caspases are vital to apoptosis and exist in the cell as inactive zymogens. Dimerization is central to procaspase activation because the active sites are comprised of loops from both monomers. Although initiator procaspases are stable monomers until activated on cell death scaffolds, the effector caspases, such as procaspase-3, are stable dimers.