Current Protein and Peptide Science - Volume 10, Issue 6, 2009

Protein phosphorylation is a common signaling mechanism in both prokaryotic and eukaryotic organisms. Whilst serine, threonine and tyrosine phosphorylation dominate much of the literature there are several other amino acids that are phosphorylated in a variety of organisms. Two of these phosphoamino acids are phosphoarginine and phospholysine. This review will focus on the chemistry and biochemistry of both phosphoarginine and phospholysine. In particular we focus on the biological aspects of phosphoarginine as a means of storing and using metabolic energy (in place of phosphocreatine in invertebrates), the chemistry behind its synthesis and we examine the chemistry behind its highenergy phosphoramidate bond. In addition we will be reporting on the incidence of phosphoarginine in mammalian cells. Similarly we will be reviewing the current findings on the biology and the chemistry of phospholysine and its involvement in a variety of biological systems.

The platelet glycoprotein receptor regulates the adhesion of blood platelets to damaged blood vessel walls and the subsequent platelet aggregation. One of the subunits, platelet glycoprotein Ibα (GpIbα), binds thrombin, a serine protease with both procoagulant and anticoagulant activities. Two groups reported the crystal structures of the complex between thrombin and the N-terminal extracellular domain (leucine-rich repeat [LRR] domain) of GpIbα. In both these structures, GpIbα was reported to bind two thrombin molecules, but both the primary and secondary thrombin binding sites differed between them. We performed a detailed comparison of the two structures to look for insights that may explain the differences. Our results show that the 1:1 GpIbα-thrombin complex detected in solution between the crystallized proteins is likely the only strong interaction. The anionic sequence (residues 268-284) of GpIbα is likely responsible for the initial interaction with thrombin and the interaction with the rest of LRR domain of GpIbα occurs subsequently and may alternate between two or more different binding modes. Our modelling suggests the interaction between GpIbα and thrombin is highly pH-dependent and a small change in pH is likely to contribute to the formation of alternate binding modes. The differences in the crystal structures reported for the GpIbα-thrombin complex suggest a fascinating plasticity in this protein-protein interaction that may be biologically significant.

The concept of Receptor Mosaic (RM) is discussed; hence the integrative functions of the assemblage of Gprotein coupled receptors physically interacting in the plane of the plasma membrane. The main focus is on a heterotrimer of G-protein coupled receptors, namely the A2A-D2-CB1 receptor trimer. A bioinformatics analysis was carried out on the amino acid sequence of these receptors to indicate domains possibly involved in the receptor-receptor interactions. Such a bioinformatic analysis was also carried out on the RM formed by mGLU R5, D2 and A2A. The importance of topology, i.e., of the reciprocal localisation of the three interacting receptors in the plan of the membrane for the RM integrative functions is underlined. However, it is also pointed out that this fundamental aspect still waits techniques capable of an appropriate investigation. Finally, it is discussed how RM topology can give hints for a structural definition of the concept of hub receptor. Thus, just as in any network, the receptor operating as a hub is the one that in the molecular network formed by the receptors has the highest number of inputs.

The Mastermind-like (MAML) proteins are transcriptional coactivators for Notch signaling, an evolutionarily conserved pathway that plays several key roles in development and in human disease. The MAML family contains MAML1, MAML2, and MAML3. More recently, MAML1 has been shown to function as a coactivator for the tumor suppressor p53, the MADS box transcription enhancer factor (MEF) 2C, and β-catenin. In addition, MAML1 has been reported to interact with the histone acetyltransferase p300, and this interaction increases p300 activity. Furthermore, MAML1 binds to CDK8, which is a subunit of the Mediator complex. The function of MAML1 as a coactivator for diverse activators, and MAML1 interaction with broadly used coactivators, suggests that MAML1 might be a key molecule that connects various signaling pathways to regulate cellular processes in normal cells and in human disease.

Class-I cytokines represent a large group of molecules involved in different physiological processes including host defence, immune regulation, food intake, energy metabolism and, relevant for this review, reproduction. In this latter respect, here, we focus the attention on four of these molecules, specifically leptin, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). These cytokines present similar three-dimensional fold structure, interact with related class-I receptors, which are expressed in the same regions (i.e., hypothalamus), and activate similar intracellular pathways. Leptin and CNTF share functional similarities, by acting at hypothalamic and pituitary levels, and their receptors are colocalized in the arcuate and paraventricular nuclei of the hypothalamus. For both these molecules, no effect on GnRH migration has been described. LIF has also been shown to affect gonadotropin secretion and here we present the novel observation that it is also able to stimulate GnRH secretion in vitro. Moreover, in the mouse, LIF is prenatally expressed in nasal regions where GnRH neurons originate and start their migration, and in vitro it stimulates intrinsic cell motility and directional migration. The role of the prototypical cytokine, IL-6, on the GnRH-LH axis is not fully clear and additional information seem necessary to better clarify this aspect. In conclusion, the data here discussed suggest that this family of cytokines appears to participate to the complex control of the reproductive function by affecting the development and function of the hypothalamus-pituitary system at different ontogenic times and anatomical sites.

Anionic antimicrobial peptides / proteins (AAMPs) were first reported in the early 1980s and since then, have been established as an important part of the innate immune systems of vertebrates, invertebrates and plants. These peptides are active against bacteria, fungi, viruses and pests such as insects. AAMPs may be induced or expressed constitutively and in some cases, antimicrobial activity appears to be a secondary role for these peptides with other biological activities constituting their primary role. Structural characterization shows AAMPs to generally range in net charge from -1 to -7 and in length from 5 residues to circa 70 residues and for a number of these peptides, post-translational modifications are essential for antimicrobial activity. Membrane interaction appears key to the antimicrobial function of AAMPs and to facilitate these interactions, these peptides generally adopt amphiphilic structures. These architectures vary from the α-helical peptides of some amphibians to the cyclic cystine knot structures observed in some plant proteins. Some AAMPs appear to use metal ions to form cationic salt bridges with negatively charged components of microbial membranes, thereby facilitating interaction with their target organisms, but in many cases, the mechanisms underlying the antimicrobial action of these peptides are unclear or have not been elucidated. Here, we present an overview on current research into AAMPs, which suggests that these peptides are an untapped source of putative antimicrobial agents with novel mechanisms of action and possess potential for application in the medical and biotechnological arenas.

Conformational flexibility is an inherent property of the protein structure. Large scale changes in the protein conformation play a key role in a variety of fundamental biological activities and have been implicated in a number of diseases. The time scales of functionally relevant dynamic processes in proteins generally do not allow the researchers to study them by the means of detailed atomic level simulations. Therefore, less computationally demanding methods based on the coarse grained models of protein structure and bioinformatics approaches are particularly important for the flexibility- related studies. This review is focused on two broad categories of protein flexibility - protein disorder and conformational switches. In the case of protein disorder, a flexible protein segment or entire protein is structurally disordered, meaning that it does not have a well-defined folded 3D structure. In the case of conformational switches, the protein backbone of a flexible segment can change or “switch” from one specific folded 3D conformation to another. In this review, the relative strengths and limitations of the existing computational tools, mostly from the bioinformatics domain, used to study and predict protein disorder and conformational switches will be discussed and the main challenges will be highlighted.

There has been a tremendous increase in the number of approved drugs derived from recombinant proteins; however, their development as potential drugs has been hampered by their instability that causes difficulty to formulate them as therapeutic agents. It has been shown that the reactivity of thiol and disulfide functional groups could catalyze chemical (i.e., oxidation and beta-elimination reactions) and physical (i.e., aggregation and precipitation) degradations of proteins. Because most proteins contain a free Cys residue or/and a disulfide bond, this review is focused on their roles in the physical and chemical stability of proteins. The effect of introducing a disulfide bond to improve physical stability of proteins and the mechanisms of degradation of disulfide bond were discussed. The qualitative/quantitative methods to determine the presence of thiol in the Cys residue and various methods to derivatize thiol group for improving protein stability were also illustrated.