Current Protein and Peptide Science - Volume 3, Issue 6, 2002

The regulation of the hepatic glucose output through glycogenolysis is an important target for type 2 diabetes therapy. Glycogenolysis is catalyzed in liver, muscle and brain by tissue specific isoforms of glycogen phosphorylase (GP). Because of its central role in glycogen metabolism, GP has been exploited as a model for structureassisted design of potent inhibitors, which may be relevant to the control of blood glucose concentrations in type 2 diabetes. Several regulatory binding sites have been identified in GP, such as the catalytic, the allosteric, and the inhibitor binding sites. Protein crystallography has contributed significant structural information on the specificity and interactions that distinguish the binding sites, and also revealed a new unexpected binding site (new allosteric site). In this review, the kinetic, crystallographic binding, and physiological studies of a number of compounds, inhibitors of GP, are described, and the essential inhibitory and binding properties of specific compounds are analyzed in an effort to provide rationalizations for the affinities of these compounds and to exploit the molecular interactions that might give rise to a better inhibitor. These studies have given new insights into fundamental structural aspects of the enzyme enhancing our understanding of how the enzyme recognizes and specifically binds ligands, that could be of potential therapeutic value in the treatment of type 2 diabetes.

It is possible that enkephalins are involved in the pain-modulating mechanism in the spinal cord. Enkephalins, however, are short-lived, being rapidly degraded by various endogenous enzymes. Many substances that inhibit enkephalin-degradation have been investigated and it has been reported that some inhibitors (e.g. kelatorphan and RB101) alone showed anti-nociceptive activity.We found an endogenous factor that modulated enkephalin-degrading activity and purified it from bovine spinal cord based on its inhibitory activity toward enkephalin-degrading enzymes. Structural analysis revealed the factor to be Leu- Val-Val-Tyr-Pro-Trp-Thr and it was named spinorphin. It has been found that spinorphin inhibited the activity toward various enkephalin-degrading enzymes from monkey brain, especially dipeptidyl peptidase III (DPPIII, Ki=5.1 x 10 -7 M). Recently we reported that this inhibitor significantly inhibited bradykinin (BK)-induced nociceptive flexor responses. Importantly, the mode of inhibition to BK-responses by spinorphin was different from the case with morphine. The morphine-induced blockade of BK-response was attenuated by pertussis toxin treatment, whereas that of spinorphin was not.We also have reported roles for spinorphin in inflammation. Spinorphin significantly inhibited the functions of polymorphonuclear neutrophils (PMNs) by suppressing the binding of fMLF to its receptor on PMNs. Further, this inhibitor suppressed the carrageenan-induced accumulation of PMN in mouse air pouches after intravenous administration. These results indicate that spinorphin may be an endogenous anti-inflammatory regulator. The possible role of spinorphin and its analog as regulators in pain and inflammation will be discussed.

Protein electrostatic properties stem from the proportion and distribution of polar and charged residues. Polar and charged residues regulate the electrostatic properties by forming shortrange interactions, like salt-bridges and hydrogen-bonds, and by defining the over-all electrostatic environment in the protein. Electrostatics play a major role in defining the mechanisms of proteinprotein complex formation, molecular recognitions, thermal stabilities, conformational adaptabilities and protein movements. For example:- Functional hinges, or flexible regions of the protein, lack short-range electrostatic interactions, Thermophilic proteins have higher electrostatic interactions than their mesophilic counter parts, Increase in binding specificity and affinity involve optimization of electrostatics, High affinity antibodies have higher, and stronger, electrostatic interactions with their antigens, Rigid parts of proteins have higher and stronger electrostatic interactions.In this review we address the significance of electrostatics in protein folding, binding and function. We discuss that the electrostatic properties are evolutionally selected by a protein to perform an specific function. We also provide bona fide examples to illustrate this. Additionally, using continuum electrostatic and molecular dynamics approaches we show that the “hot-spot” inter-molecular interactions in a very specific antibody-antigen binding are mainly established through charged residues. These “hot-spot” molecular interactions stay intact even during high temperature molecular dynamics simulations, while the other inter-molecular interactions, of lesser functional significance, disappear. This further corroborates the significance of charge-charge interactions in defining binding mechanisms. High affinity binding frequently involves “electrostatic steering”. The forces emerge from over-all electrostatic complementarities and by the formation of charged and polar interactions. We demonstrate that although the high affinity binding of barnase-barstar and anti-hen egg white lysozyme (HEL) antibody-HEL complexes involve different molecular mechanisms, it is electrostatically regulated in both the cases. These observations, and several other studies, suggest that a fine tuning of local and global electrostatic properties are essential for protein binding and function.

Newly synthesized proteins have an intrinsic signal sequence, functioning as “address tags” or “zip codes”, that is essential for guiding them wherever they are needed. Owing to such a unique function, protein signals have become a crucial tool in finding new drugs or reprogramming cells for gene therapy. However, to effectively use protein signals as a desirable vehicle in the field of proteomics, the first important thing is to find a fast and powerful method to identify the “address tag” or “zip code” entity. Although all signal sequences contain a hydrophobic core region, they show great variation in both overall length and amino acid sequence. It is this variation that makes it possible to deliver thousands of proteins to many different cellular locations by varieties of modes. It is also this variation that makes it very difficult to formulate a general algorithm to predict signal sequences. Nevertheless, various prediction models and algorithms have been developed during the past 17 years. This Review summarizes the development in this area, from the pioneering methods to neural network approaches, and to the sub-site coupling approaches. Meanwhile, the future challenges in this area, as well as some promising avenues for further improving the prediction quality, have been briefly addressed as well.

Inflammation, a localized response to tissue injury, and disorders characterized by inflammation are difficult problems in clinical medicine. This difficulty stems in large part from incomplete understanding of inflammatory processes and their regulation. Recent development of knowledge of the role of central nervous system and neuroendocrine system in host responses has provided a new view of the capacity of neuronal and soluble mediators in these systems to influence inflammation. One of these mediators is the endogenous neuropeptide α-melanocyte stimulating hormone (α-MSH), which is an N-acetyl tridecapeptide derived from the cleavage of a larger precursor molecule, pro-opiomelanocortin (POMC).α-MSH is widely distributed in tissues of higher organisms, it has been identified in the pituitary, various brain regions, skin, circulation and other sites. The neuropeptide α-MSH is important to the natural limitation of fever, which is an early host response to endotoxin. In addition to its action within the brain to reduce fever, α-MSH has potent and broad antiinflammatory effects in many forms of inflammation. This review will summarize the data on the actions of the peptide on various aspects of peripheral and central inflammation. On the basis of the data presented, we may think that the antiinflammatory actions of the peptide via peripheral and / or central melanocortin receptors might put the peptide into practice therapeutically in near future.

The native structures of proteins are governed by a large number of non-covalent interactions yielding a high specificity for the native packing of structural elements. This allows for the reconstitution of proteins from disconnected polypeptide fragments. The specificity for the native arrangement also enables interchange of structural elements with another identical protein chain resulting in dimers with swapped segments. Proteins are not static structures, but open up repetitively on a timescale of minutes to years depending on the identity of the protein and solution conditions. The open protein may self-close and return to the native state, or it may close with another polypeptide chain leading to 3D domain swapping. The term describes two or more protein molecules swapping identical domains or smaller secondary structure elements. The non-covalent intra-molecular interactions between domains in the monomer are thus broken and restored in the oligomer by identical inter-molecular contacts. This review will discuss 3D domain swapping in relation to protein reconstitution and fibril formation. Examples of reconstituted and domain-swapped proteins will be given. The physiological benefits of 3D domain swapping will be discussed, as well as its role in the evolution of proteins and pathology.

Prions are a novel class of infectious pathogens that cause a group of fatal prion diseases in which the benign cellular form of the prion protein (PrP C) is transformed into the disease-related scrapie variant (PrP SC). The two PrP isoforms differ in their structure and resistance to degradation. The molecular mechanism by which the PrP SC is formed and causes infectivity or neurodegeneration is not known. In a compelling and emerging view, posttranslational modifications (or the lack thereof) play roles in the transformation of PrP C to PrP SC. Human PrP contains two consensus sites for N-linked glycosylation, at Asn181 and Asn197. From the functional standpoint, glycosylation can modify either the conformation of PrP C, or the stability of PrPSC and, hence, the rate of PrPSC clearance. So far the NMR structures of only recombinant, non-glycosylated prions are known, while the structure of the glycosylated form is estimated by molecular modeling. A number of native amino acid mutations in PrP can be mapped near the glycosylation sites. Normal prion protein has been demonstrated to be a copper binding protein, and increasing evidence has shown correlation between the level of PrP expression and tolerance to oxidative stress. Moreover, histochemistry for nitrotyrosine is used for detection of neuronal labeling, a sign of a peroxynitrite-mediated neuronal degradation and a marker for nitrative stress in scrapie-infected mouse brains. It is an intriguing proposition that the post translational modifications alone, or in combination with amino acid changes, play dominant roles in the pathogenic transformation of PrPC to PrPSC.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells.Pgp was found abnormally expressed on the plasma membrane of tumor cells with “classical” MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100).Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.