Current Protein and Peptide Science - Volume 5, Issue 4, 2004

Life on our planet has adapted to a wide range of physical conditions, including extremely high and low temperatures, high pressures, extremes of pH and chemically aggressive conditions. To cope with these stress factors, organisms have evolved a variety of strategies operating on very different levels, from the small molecule response to physiological and behavioural adaptation. The only kind of stress response that is found universally in all species is the stress-induced expression or overexpression of specific proteins. Among these, the heat shock proteins are the best studied group. They have been shown to serve in a variety of specific functions, including those of molecular chaperones, proteases, and “capacitors of evolution.” An overview of these different functions and also of the other kinds of stress proteins is given, with a perspective on how they serve the survival of the cell and the species in the presence of environmental stress factors, and how they can be used in medical applications.

In spite of the relevance of the results obtained through the clinical application of chemotherapeutic agents (reverse transcriptase and proteinase inhibitors) that are able to prolong the life span of affected people, acquired immunodeficiency syndrome (AIDS) remains a serious and lethal disease. AIDS is caused by a type 1 human immunodeficiency virus (HIV-1) and formation of a complex among the gp120, CD4 and CCR5 / CXCR4 surface proteins represents a key-step in the infection. The use of synthetic peptides reproducing reduced sequences of these proteins has contributed to increase the knowledge of the mechanism that determines the penetration of the HIV viruses into the targetcells. In addition, short peptides with minimum structural requirements for anti-HIV activity hold greater potential as lead compounds for rational drug design than macromolecular proteins. In this context, our studies concern: the role of gp120 V3 loop in CD4 binding, the importance of the N-terminal sequence of HIV CCR5 coreceptor, the potential inhibitory properties of sequences patterned on CXCR4 natural ligand (SDF-1) and the role of secondary structure in determining gp160 enzymatic processing into gp120.

Understanding the interactions between activating or antagonizing ligands and their cognate receptors at a molecular level offers promise for the development of pharmacological therapeutics for CNS disorders. The discovery of novel molecules that are capable of discriminating between the varied molecular subunits or isoforms of ion channels should provide a more detailed understanding of the pathophysiology of many CNS disorders. Abundant natural sources of pharmacologically active agents that demonstrate this refined selectivity and specificity are found in the animal toxins of venomous species including: snakes, spiders and the marine snail of the genus Conus. The uniquely fascinating combinatorial ability of the marine snail, genus Conus to modify the pharmacological properties of these neurotoxins or conopeptides within its venom is depicted throughout this review. The myriad of posttranslational modifications and disulfide bonded architectures that have been identified in the conopeptides, are described with an emphasis on the unique pharmacological properties and receptor target specificities that have been ascribed to each of these modifications. The ability of NMR spectroscopy to provide three-dimensional structural information within the interaction interface for both the ligand and target protein following complex formation and its application to conopeptide drug discovery are discussed. Similarly, the strength of merging NMR spectroscopy data with ab initio “;restrained soft-docking” for rational pharmacophore design and the identification of lead compounds from in silico library screens will also be discussed. The initial phases of this stratagem are illustrated using two toxin antagonists and the recently determined structure of the KcsA potassium channel. These data exemplify the utility of this approach in elucidating important molecular interfaces of specific toxin-receptor / ion channel complexes, which can be further exploited in drug discovery initiatives.

Modern protein secondary structure prediction methods are based on exploiting evolutionary information contained in multiple sequence alignments. Critical steps in the secondary structure prediction process are (i) the selection of a set of sequences that are homologous to a given query sequence, (ii) the choice of the multiple sequence alignment method, and (iii) the choice of the secondary structure prediction method. Because of the close relationship between these three steps and their critical influence on the prediction results, secondary structure prediction has received increased attention from the bioinformatics community over the last few years. In this treatise, we discuss recent developments in computational methods for protein secondary structure prediction and multiple sequence alignment, focus on the integration of these methods, and provide some recommendations for state-of-the-art secondary structure prediction in practice.

The peptide hormone angiotensin II is well established to play an endocrine role in the regulation of blood pressure, fluid and electrolyte homeostasis. In addition to its hemodynamic function, recent studies have shown that numerous tissues and organs contain their own locally generated angiotensin products (angiotensin II, III, IV and Ang 1-7) and they exhibit their respective activities. Such an intrinsic angiotensin-generating system renders to specific tissue function of our body, frequently via the regulatory mechanism of a paracrine, autocrine or intracrine manner. These tissues and organs include, to name but a few, the brain, bone marrow, adipose, epididymis, carotid body, liver, and pancreas. This local system has been shown to be responsive to various stimuli of physiological and pathophysiological importance. Moreover, the locally generated angiotensin peptides have multiple and novel actions including cell growth, anti-proliferation, apoptosis, reactive oxygen species generation, hormonal secretion, pro-inflammatory, and pro-fibrogenic actions, as well as vasoconstriction and vasodilatation. Notwithstanding the emerging roles of angiotensin II in various tissues and organs, the physiological significance and ultimately the clinical relevance remain largely undefined. Future target for these new functions by making use of specific renin-angiotensin system inhibitors, such as the angiotensin-converting enzyme and angiotensin II receptor blockers either in mono-therapy or in combination, could be of clinical importance. The current review is to focus on some of the new functions arising from the locally formed angiotensin II in tissues and organs, with particular attention to its emerging roles in the liver and the pancreas.

The fact that cleavage of single peptide linkages in proteins often leads to extensive conformational alteration, including regions far removed from the cleavage site is not fully understood. We propose, based on the work of Linderstrom-Lang and Schellman, that disruption primarily occurs within protein structural domains that are stabilized by cooperative interactions and that cleavage of single peptide linkages of the domain perturbs the entire cooperative interaction. For this model we review experimental observations: on fragment complexation (ribonuclease A, staphylococcal nuclease and cytochrome c), destabilized N-terminal large fragments (ribonuclease A and nuclease), cooperative folding and stabilization of proteins (ribonuclease A, nuclease and cytochrome c), the close relationship of the three-dimensional structure between fragment complexes and the original protein (ribonuclease A and nuclease), ligand induced stabilization (nuclease), 3D domain swapping, circular permutation (dihydrofolate reductase), evolutionary conservation (cytochrome c fold). Based on analysis of these observations, we conclude that the cooperative interactions of domains are important for the mechanism of 3D domain swapping as well as for stabilization and thereby, determination of the ground state of native proteins. Furthermore, analysis of the observations reveals that domains generally contain a hydrophobic core. Further, based on studies of cytochrome c and the Tsao, Evans and Wennerstrom model of electrostatic interactions between two hydrophobic monolayers, we propose the model that the hydrophobic core of a domain is polarizable and responds to the surface charges through its polarizability to stabilize the domain, explaining in part the nature of the cooperative interactions.

Most proteins function as multiprotein complexes or interact with multiprotein complexes. Identification of protein-protein interactions in the context of their physiologically relevant complexes is therefore key to fully understand the cellular machinery. Here I discuss advances in chemical crosslinking methods that allow investigators to map direct subunit contacts in transient interactions with multimeric complexes. Methods discussed fall into two categories: (i) in vitro approaches with localized, inducible crosslinking reagents and (ii) in vivo approaches with unlocalized crosslinkers.