Current Protein and Peptide Science - Volume 3, Issue 4, 2002

Protein tyrosine phosphatases, SH2 and PTB domains are crucial elements for cellular signal transduction and regulation. Much effort has been directed towards elucidating their specificity in the past decade using a variety of approaches. Combinatorial library methods have contributed significantly to the understanding of substrate and ligand specificity of phosphoprotein recognizing domains.This review gives a brief overview of the structural characteristics of protein tyrosine phosphatases, SH2 and PTB domains and their binding to phosphopeptides. The chemical synthesis of peptides containing phosphotyrosine or phosphotyrosine mimics and the various formats of synthesis and deconvolution of combinatorial libraries are explained in detail. Examples are given as how different combinatorial libraries have been used to study the interaction of phosphopeptides with SH2 domains and phosphatases. The intrinsic advantages and difficulties of library synthesis, screening and deconvolution are pointed out. Finally, some experimental results on the substrate specificity of protein tyrosine phosphatase 1B and the SH2 domain of the adaptor protein Grb-2 are summarized and discussed.

Most patients with cancer are treated with chemotherapy but die from progressive disease or toxicities of therapy. Current chemotherapy regimens primarily use cytotoxic drugs which damage cell DNA or impair cell proliferation in both malignant and normal tissues. After several treatment courses, the patients' tumor cells often overexpress multi-drug resistance genes which prevent further tumor cytoreduction. Novel agents which can kill such resistant tumor cells are needed. One such class of agents are targeted peptide toxins. Targeted peptide toxins consist of peptide toxins covalently linked to tumor selective peptide ligands. These molecules bind tumor cell surface receptors, internalize, and facilitate transfer of the toxin catalytic domains to the cytosol. Once in the cytosol, the enzyme activity leads to cell death. A number of plant, bacterial and fungal toxins have been used, and clinical trials with several of these have produced complete remissions in chemoresistant neoplasms. Nevertheless, there is a continuing need for novel targeted toxins. Many patients have pre-existing antibodies against the currently clinically used toxins and many toxins are inactive when used for myeloid malignancies where internalized proteins are rapidly routed and degraded in lysosomes. Anthrax toxins are the cytotoxic components of Bacillus anthracis. While the bacteria has been the source of serious illness, deaths and global anxieties related to past or future bioterrorism, the isolated toxins do not pose public health hazards. In fact, toxin treated patients will likely develop protective antibodies. Anthrax toxin is an excellent choice for tumor cell surface targeting. Other than U.S. military personnel immunized during the Gulf War, most people lack preexisting antibodies. This may change in the future due to threats of additional terrorist acts, but for the present few patients will have antibodies to anthrax proteins. The separate subunits for binding, translocation and cell killing facilitate genetic engineering to yield tumor-specific cell killing. The toxins are more potent than most of the other peptide toxins and may yield highly efficacious targeted molecules. This essay will review anthrax toxin structure-function, preliminary experiments with re-targeted anthrax toxin and potential designs for new ligand-anthrax therapeutics.

Matrix metalloproteinases (MMPs) are a pivotal family of zinc enzymes responsible for degradation of the extracellular matrix (ECM) components including basement membrane collagen, interstitial collagen, fibronectin, and various proteoglycans, during normal remodeling and repair processes. The potent proteolytic activities of MMPs is mainly regulated by the balance with specific tissue inhibitors of Matrix metalloproteinases (TIMP). Excessive or inappropriate expression of MMP may contribute to the pathogenesis of tissue destructive processes in a wide variety of diseases including lung diseases. Although the precise mechanisms are still unknown, the contribution of individual MMPs are worth investigating in seeking the pathogenesis of various lung diseases such as lung cancer, bronchial asthma, chronic obstructive pulmonary disease, acute lung injury, pulmonary hypertension and interstitial lung diseases. In particular, the close association of each lung disease with the destructive effects of gelatinase A and B (also called MMP-2 and MMP-9) on the basement membrane in early alveolar remodeling, and that of collagenase-1 (MMP-1) on the major interstitial structural protein of ECM have received considerable attention. The interaction of MMPs with chemical mediators and inflammatory cytokines has also been reported in some recent studies.Several promising therapeutic approaches to inhibit MMPs have just started in the field of oncology, while more specific MMP inhibitors may be required for further investigation in other fields of lung diseases. In this review, The main focus is on the recent clinical and experimental findings and the contributions of MMPs and / or TIMPs in the lung diseases.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from the ovine brain in 1989 as a novel hypothalamic hormone that potently activates adenylate cyclase to produce cyclic AMP in pituitary cells. This neuropeptide belongs to the secretin / glucagon / vasoactive intestinal peptide (VIP) superfamily, and exists in two amidated forms as PACAP38 (38-amino acid residues) and PACAP27 derived from the same precursor. The primary structure of PACAP has been remarkably conserved throughout evolution among tunicata, ichthyopsida, amphibia and mammalia, and a PACAP-like neuropeptide has also been determined in Drosophila. Both PACAP and its receptors are mainly distributed in the nervous and endocrine systems showing pleiotropic functions with high potency. There are three types of receptors with high PACAP-binding affinity and with different tissue-distribution patterns. All of them belong to G-protein-coupled receptor superfamily with seven transmembrane domains. PAC1 is the PACAP-specific receptor and exists in at least eight splice variants which couple to different intracellular signal transduction pathways. VPAC1 and VPAC2 are the common receptors for both PACAP and VIP, which are coupled to adenylate cyclase. This review article presents and discusses an update on PACAP research and its pleiotropic physiological functions based on multiple receptor-mediated signaling mechanisms in both the central and peripheral nervous system, including the regulation of hypothalamic neurosecretion, homeostatic control of circadian clock and behavioral actions, involvement in learning and memory processes, neuroprotective effects such as anti-apoptosis and response to injury and inflammation, and neural ontogenetic functions on proliferation / differentiation processes from early stages.

Quantitation of protein 3-D structure similarity is crucial in such fields as evolutionary studies, structural modeling and prediction of biological function. There are various approaches, many of which are tailored to specific problems. This review summarizes the recent developments in this field with particular interest in two main areas: i) improvements to and statistical interpretation of the root-man-square distance between equivalent atoms, rmsd, and ii) methods of protein structural classification based on geometrical features. Special attention is given to fast methods capable of analyzing large structural databases.

The mitochondrial F1Fo ATP synthase complex has a key role in cellular energy metabolism. The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F1, protruding out of the inner side of the membrane, a membrane integral proton translocating moiety Fo, and a stalk connecting F1 to F o. The Xray crystallographic analysis of the structure of the bovine mitochondrial F1 ATPase has provided a structural basis for the binding-change rotary mechanism of the catalytic process in F1, in which the γ subunit rotates in the central cavity of the F1 α3 / β3 hexamer. Rotation of γ and ε subunits in the E. coli enzyme and of, γ and δ subunits in the mitochondrial enzyme, is driven, during ATP synthesis, by proton motive rotation of an oligomer of c subunits (10-12 copies) within the Fo base piece. Average analysis of electron microscopy images and cross-linking results have revealed that, in addition to a central stalk, contributed by γ and δ / ε subunits, there is a second lateral one connecting the peripheries of Fo and F1.To gain deeper insight into the mechanism of coupling between proton translocation and catalytic activity (ATP synthesis and hydrolysis), studies have been undertaken on the role of F1 and Fo subunits which contribute to the structural and functional connection between the catalytic sector F1 and the proton translocating moiety Fo. These studies, which employed limited proteolysis, chemical cross-linking and functional analysis of the native and reconstituted F1Fo complex, as well as isolated F1, have shown that the N-terminus of α subunits, located at the top of the F1 hexamer is essential for energy coupling in the F1Fo complex. The α N-terminus domain appears to be connected to Fo by OSCP (Fo subunit conferring sensitivity of the complex to oligomycin). In turn, OSCP contacts FoI-PVP(b) and d subunits, with which it constitutes a structure surrounding the central γ and δ rotary shaft.Cross-linking of FoI-PVP(b) and γ subunits causes a dramatic enhancement of downhill proton translocation decoupled from ATP synthesis but is without effect on ATP driven uphill proton transport. This would indicate the existence of different rate-limiting steps in the two directions of proton translocation through Fo.In mitochondria, futile ATP hydrolysis by the F1Fo complex is inhibited by the ATPase inhibitor protein (IF1), which reversibly binds at one side of the F1Fo connection. The trans-membrane ΔpH component of the respiratory Δp displaces IF1 from the complex, in particular the matrix pH is the critical factor for IF1 association and its related inhibitory activity. The 42L-58K segment of the IF1 has been shown to be the most active segment of the protein, it interacts with the surface of one α / β pairs of F1, thus inhibiting, with the same pH dependence as the natural IF1, the conformational interconversions of the catalytic sites involved in ATP hydrolysis.IF1 has a relevant physiopathological role for the conservation of the cellular ATP pool in ischemic tissues. Under these conditions IF1, which appears to be over expressed, prevents dissipation of the glycolytic ATP.

The exact mechanism by which four Fe-Protoporphyrin-IX (heme) moieties and four nascent globin chains combine to form human hemoglobin (α2β2) remains a mystery. Recent Soret spectral static and kinetic studies of the incorporation of CN-Hemin derivatives into an array of human globin species have provided in vitro evidence of an ordered assembly pathway, through an αheme-βglobin intermediate, that ensures correct formation of active hemoglobin tetramers.

A variety of factors contribute to the complex course of inflammation. Microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. In the early phase of inflammation, excessive amounts of cytokines and inflammatory mediators are released. These factors activate, in addition to other signaling pathways, the lipid synthesis pathways, which play a crucial role in the pathogenesis of organ dysfunction. Arachidonic acid (AA), the precursor of pro-inflammatory eicosanoids, is released from membrane phospholipids by the action of phospholipase A2 (PLA2), and is metabolized to prostaglandins (PGs) and leukotrienes (LTs) by the action of cyclooxygenase (COX) and lipoxygenase (LO) enzymes, respectively. Disordered activation of PLA2, LO and COX enzymes have been implicated in many inflammatory diseases. PLA2 is activated by phospholipase-A2-activating protein (PLAP) and LO by 5-lipoxygenase-activating protein (FLAP). The inducible form of COX-2 enzyme, which is usually not present under basal conditions, is induced in inflammation. In this article the function of these enzymes in eicosanoid synthesis, their regulation, and their implication in inflammatory disorders will be reviewed. The properties, function and regulation of the protein activators PLAP and FLAP will also be discussed.