Phospholipase A2Mechanism Of Interfacial Activation,An Interdiscliplinary Approach

Phospholipase A2 (PLA2) is an enzyme that hydrolyzes the sn-2-ester bond of membrane phospholipids and liberates arachidonic acid, which is converted to eicosanoids that act as potent mediators of inflammation and allergy. As such this enzyme plays a crucial role in many homeostatic physiological and immunologic processes and disease progression. PLA2s undergo substantial increase in activity upon binding to cellular membranes. This effect of interfacial activation is well recognized, yet its structural and physical aspects are poorly understood. In this work, we have employed the interdisciplinary methods of molecular biology, biochemistry, biophysics, bioinformatics and computational biology, in order to elucidate the structure-function relationships mediating the interfacial activation of human group IIA and group IB PLA2 isoforms. We have evaluated the structural and functional consequences of two conservative, single residue substitutions, located at key membrane-binding and substrate-binding positions of hIIA PLA2. We have also evaluated a human group IB fragment (hIBΔN10), missing the first 10 N-terminal residues which make up the N-terminal alpha helix, as well as a chimeric enzyme substituting the N-terminal alpha helix of hIB PLA2 with that from hIIA PLA2 (hIIA/IB PLA2). We have compared the engineered proteins against both the hIIA and hIB PLA2 native enzymes and their N-terminal peptides, N10-hIB and N10-hIIA, respectively. We have developed and used a novel multidisciplinary approach in order to position the segmentally labeled hIB PLA2 and hIIA/IB chimeric PLA2s at the membrane surface. The results of this work provide significant insight into the understanding of the physical aspects of interfacial activation by determining the precise membrane binding modes of PLA2 isoforms and identifying certain amino acid residues and whole protein segments that play key roles in membrane binding, activation, and involved allosteric conformational effects in PLA2s

A novel mode of translocation for cytolethal distending toxin

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB. (c) 2008 Elsevier B.V. All rights reserved

Evidence for the regulatory role of the N-terminal helix of secretory phospholipase A(2) from studies on native and chimeric proteins

The phospholipase A(2) (PLA(2)) enzymes are activated by binding to phospholipid membranes. Although the N-terminal alpha-helix of group I/II PLA(2)s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA(2)s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA(2), we have created semisynthetic human group IB PLA(2) in which the N-terminal decapeptide is joined with the C-13-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA(2) joined with a C-13-labeled fragment of group IB PLA(2). Infrared spectral resolution of the unlabeled and C-13-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA(2) has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA(2) is more rigid than that of the IB PLA(2), but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA(2), which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA(2). Correlation is delineated between structural and membrane binding properties of PLA(2)s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA(2)s acts as a regulatory domain that mediates interfacial activation of these enzymes

Therapeutic Modulation of Apoptosis: Targeting the BCL-2 Family at the Interface of the Mitochondrial Membrane

A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease

Transmembrane Pore Formation By The Carboxyl Terminus Of Bax Protein

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal helix 9 of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide-peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents. © 2012 Elsevier B.V. All rights reserved

The N-Terminal Α-Helix Of Pancreatic Phospholipase A2 Determines Productive-Mode Orientation Of The Enzyme At The Membrane Surface

Phospholipase A2 (PLA2) hydrolyzes glycerophospholipids to free fatty acid and lyso-phospholipid, which serve as precursors for the biosynthesis of eicosanoids and other lipid-derived mediators of inflammation and allergy. PLA2 activity strongly increases upon binding to the surface of aggregated phospholipid. The N-terminal ∼ten residue α-helix of certain PLA2 isoforms plays important roles in the interfacial activation of the enzyme by providing residues for membrane binding of PLA2 and by contributing to the formation of the substrate-binding pocket. However, the relative contributions of the N-terminal α-helix and the rest of the protein in membrane binding of PLA2 and its productive-mode orientation at the membrane surface are not well understood. Here we use a variety of biophysical approaches to determine the role of the N-terminal helix in membrane binding strength, orientation, and activity of human pancreatic PLA2. While the full-length PLA 2 binds to membranes with a defined orientation, an engineered PLA2 fragment ΔN10 that lacks the N-terminal ten residues binds to membranes with weaker affinity and at random orientation, and exhibits ∼100-fold lower enzymatic activity compared to the full-length PLA 2, indicating the key role of the N terminus in PLA2 function. The results of polarized infrared spectroscopic experiments permit determination of the orientation of membrane-bound PLA2 and identification of its interfacial binding surface. Moreover, the full-length PLA2 demonstrates increased conformational flexibility in solution and is stabilized upon membrane binding, while the ΔN10 fragment is more rigid than the full-length PLA2 both in free and membrane-bound states. Our results suggest that the N-terminal α-helix supports the activation of PLA2 by (a) enhancing the membrane binding strength, (b) facilitating a productive-mode orientation of PLA2 at the membrane surface, and (c) conferring conformational integrity and plasticity to the enzyme. © 2004 Elsevier Ltd. All rights reserved

Chaperonin Containing Tcp-1 Protein Level In Breast Cancer Cells Predicts Therapeutic Application Of A Cytotoxic Peptide

Purpose: Metastatic disease is a leading cause of death for patients with breast cancer, driving the need for new therapies. CT20p is a peptide previously discovered by our group that displays cancer-specific cytotoxicity. To design the optimal therapeutic use of the peptide, we identified the intracellular target of CT20p in breast cancer cells, correlating expression patterns of the target with susceptibility to CT20p. Experimental Design: Using polymeric nanoparticles to deliver CT20p, we assessed cytoskeletal changes, cell migration, adhesion, and viability in cells treated with the peptide. Protein pull-down experiments, coupled to mass spectrometry, enabled identification of the peptide\u27s intracellular target. Biochemical and histologic techniques validated target identity in human cell lines and breast cancer tissue microarrays and revealed susceptibility patterns to CT20p.Results: Chaperonin containing TCP-1 (CCT) was identified as the intracellular target of CT20p. Cancer cells susceptible to CT20p had increased CCT, and overexpression of CCTb, a subunit of the CCT complex, enhanced susceptibility to CT20p. Susceptible cells displayed reduced tubulin, a substrate of CCT, and inhibition of migration upon CT20p treatment. CCTb levels were higher in invasive ductal carcinomas than in cancer adjacent tissues and increased with breast cancer stage. Decreased breast cancer patient survival correlated with genomic alternations in CCTb and higher levels of the chaperone. Conclusions: Increased CCT protein in breast cancer cells underlies the cytotoxicity of CT20p. CCT is thus a potential target for therapeutic intervention and serves as a companion diagnostic to personalize the therapeutic use of CT20p for breast cancer treatment. Clin Cancer Res; 22(17); 4366-79