Biomedical subjects

Cristina Pop

Publications and source records attributed to Cristina Pop.

6 recordsLinked to original sources

PubMed2006

Role of loop bundle hydrogen bonds in the maturation and activity of (Pro)caspase-3.

During maturation, procaspase-3 is cleaved at D175, which resides in a linker that connects the large and small subunits. The intersubunit linker also connects two active site loops that rearrange following cleavage and, in part, form the so-called loop bundle. As a result of chain cleavage, new hydrogen bonds and van der Waals contacts form among three active site loops. The new interactions are predicted to stabilize the active site. One unresolved issue is the extent to which the loop bundle residues also stabilize the procaspase active site. We examined the effects of replacing four loop bundle residues (E167, D169, E173, and Y203) on the biochemical and structural properties of the (pro)caspase. We show that replacing the residues affects the activity of the procaspase as well as the mature caspase, with D169A and E167A replacements having the largest effects. Replacement of D169 prevents caspase-3 autoactivation, and its cleavage at D175 no longer leads to an active enzyme. In addition, the E173A mutation, when coupled to a second mutation in the procaspase, D175A, may alter the substrate specificity of the procaspase. The mutations affected the active site environment as assessed by changes in fluorescence emission, accessibility to quencher, and cleavage by either trypsin or V8 proteases. High-resolution X-ray crystallographic structures of E167A, D173A, and Y203F caspases show that changes in the active site environment may be due to the increased flexibility of several residues in the N-terminus of the small subunit. Overall, the results show that these residues are important for stabilizing the procaspase active site as well as that of the mature caspase.

Amino Acid Sequence↗

PubMed2006

The apoptosome activates caspase-9 by dimerization.

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.

Apoptosis↗

PubMed2005

The nematode death machine in 3D.

Regulated apoptosis is part of the development of the nematode Caenorhabditis elegans. In a recent paper in Nature, Yan et al. (2005) describe the in vitro reconstitution of the core components of the worm apoptotic pathway. Based on a structural analysis of the complex between the death activator CED-4 and the antiapoptotic protein CED-9, the authors explain the regulation of activity of CED-4. Intriguingly, CED-4 comprises a AAA+ type ATPase domain yet does not seem to need ATP hydrolysis for activity.

Animals↗

PubMed2004

Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3.

We have examined the role of a salt bridge between Lys242 and Glu246 in loop L4 of procaspase 3 and of mature caspase 3, and we show that the interactions are required for stabilizing the active site. Replacing either of the residues with an alanine residue results in a complete loss of procaspase 3 activity. Although both mutants are active in the context of the mature caspase 3, the mutations result in an increase in K(m) and a decrease in kcat when compared with the wild-type caspase 3. In addition, the mutations result in an increase in the pK(a) value associated with a change in kcat with pH, but does not affect the transition observed for Km versus pH. The mutations also affect the accessibility of the active-site solvent as measured by tryptophan fluorescence emission in the presence of quenching agents and as a function of pH. We show that, as the pH is lowered, the (pro)caspase dissociates, and the mutations increase the pH-dependent instability of the dimer. Overall, the results suggest that the contacts lost in the procaspase as a result of replacing Lys242 and Glu246 are compensated partially in the mature caspase as a result of new contacts that are known to form on zymogen processing

Alanine↗

PubMed2003

An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3.

We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.

Base Sequence↗

PubMed2003

Mutations in the procaspase-3 dimer interface affect the activity of the zymogen.

The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of approximately 60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 A from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.

Base Sequence↗