Advances in Protein and Peptide Sciences

Amphitropic proteins are soluble, globular proteins that may − under certain conditions − interact reversibly with a plasma membrane. How this apparent duality in the properties of a protein is achieved has been a relatively little-studied subject until recently. In this review we aim to summarize the current knowledge regarding some important amphitropic systems in which the interaction with the membrane does not require post-translational functional groups, but is an intrinsic property of the protein. We discuss mechanisms and driving forces involved in membrane binding in the context of two related concepts in protein folding and function that appear to have implications for understanding the association of proteins with membranes. First, the existence of some proteins with low-energy barrier heights for protein folding. Low folding barriers and the ability of proteins to form stable molten globule states are rationales that can explain how a protein can gain access to an ensemble (or continuum) of non-native conformations that are competent membrane binders. Second, the focus on order-disorder and disorder-order transitions to explain protein function, a concept which has been mainly developed within the novel protein trinity paradigm. Here, protein function can arise from any of three thermodynamic states: a solid, crystal-like state; a dense fluid state; and an extended disordered state. Together these concepts aid to understand amphitropic mechanism and to unify interpretations of protein behaviour with respect to the degree of folding or unfolding of the membrane-bound proteins.

Membrane proteins, although constituting about one-third of all proteins encoded by the genomes of living organisms, are still strongly underrepresented in the database of 3D protein structures, which reflects the big challenge posed by this class of proteins. Novel and fundamental insights into the structure, function, assembly and interaction with lipids of membrane proteins are continuously revealed by structural biologists employing electron and x-ray approaches. To date, two structural motifs, α-helices and β-sheets, have been found in membrane proteins and interestingly these two structural motives correlate with the location: while α-helical bundles are most often found in the receptors and ion channels of plasma and endoplasmic reticulum membranes, β-barrels are restricted to the outer membrane of Gram-negative bacteria, the mitochondrial membrane and chloroplasts and represent the structural motif used by several microbial toxins to form cytotoxic transmembrane channels. The β-barrel, while being a rigid and stable motif is a versatile scaffold, having a wide variation in the size of the barrel, in the mechanism to open or close the gate and to impose selectivity on substrates. The difficulty in obtaining crystals suitable for high-resolution studies of outer membrane proteins has resulted in their under-representation in the Protein data Bank [1]. Even if the number of x-ray structures of integral membrane proteins has greatly increased in recent years, only a few of them provide information at a molecular level on how proteins interact with lipids that surround them in the membrane. The detailed mechanism of protein lipid interactions is of fundamental importance for understanding membrane protein folding, membrane adsorption, insertion and function in lipid bilayers. Both specific and aspecific interactions with lipids may participate in protein folding and assembly.

Membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Many of these proteins are implicated in human diseases (atherosclerosis, Alzheimer's diseases, Parkinson, cancer, and antibiotic resistance) and form excellent targets for drug discovery. Due to difficulty in protein expression, purification, reconstitution into a proper membrane model, and crystallization, however, structural determination of membrane protein has been challenging. This chapter describes recent advances in multidimensional NMR spectroscopy allowing the study of a select set of both peripheral and integral membrane proteins. Surface-binding membrane proteins discussed include amphitropic proteins, antimicrobial LL-37 and anticancer peptides, the HIV-1 gp41 peptides, human α- synuclein and apolipoproteins (A-I, A-II, C-I, C-II, C-III, and E). Also discussed are transmembrane proteins including bacterial outer membrane β-barrel proteins and oligomeric α-helical proteins. In addition, the backbone structure of human chemokine receptor CXCR1, a GPCR with seven transmembrane helices, is available via solid-state NMR studies. These structures are made possible due to reconstruction of membrane proteins in membrane-mimetic constructs such as detergent micelles, bicelles, nanodiscs, and phospholipid bilayers as well as the continued development of modern NMR technologies. In addition to protein dynamics, NMR can be applied to investigation of protein-lipid and protein-ligand interactions. These examples illustrate the unique role solution and solid-state NMR spectroscopy plays in structural biology of membrane proteins.

Host cell entry by influenza and other enveloped viruses is well characterized, however, the manner in which non-enveloped viruses deliver their genome across host cell membranes in the absence of membrane fusion remains unresolved. The discovery of short, membrane altering, amphipathic or hydrophobic sequences in several non-enveloped virus capsid proteins such as the γ (gamma) peptide of nodaviruses and tetraviruses, VP4 and the N-terminal region of VP1 of picornaviruses, μ1N of reoviruses, and protein VI of adenoviruses suggests that these small peptides facilitate breaching of the host membrane and the delivery of the viral genome into the host cell. In spite of conspicuous differences in entry among non-enveloped virions, the short stretches of membrane active regions are associated with similar, entry-related events including: i) proteolytic cleavage of a precursor capsid protein resulting in increased dynamic character and/or accessibility of these peptides; ii) structural changes in the virus capsid triggered by receptor binding and/or low pH in entry compartments, resulting in peptide exposure; iii) externalized peptides interact with host membranes and disrupt them, facilitating delivery of the viral genome inside the host cell. Here we discuss the membrane alteration activity in nonenveloped viruses with reference to the γ peptide of nodaviruses. Virtually all of the characteristics of γ are shared by analogous peptides in other non-enveloped viruses, making it a simple prototype for comparative purposes.

The degradation is critical to activation and inactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and cell death programs. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage. These exposed termini serve as unstructured initiation site or degron that target specific caspase substrates to the ubiquitin-proteasome system. Here we analyzed this hypothesis based on a list of caspase substrate proteins containing PEST motif.

The ubiquitin-mediated protein degradation pathway exerts a wide spectrum of effects and modulates a variety of biological processes including cell cycle progression, transcriptional regulation, signal transduction, antigen presentation, apoptosis (or programmed cell death), oncogenesis, preimplantation, neuron degeneration, and DNA damage repair. Recently, the importance of deubiquitination mechanism has been emerged as an essential regulatory step to control these cellular mechanisms for homeostasis. As a number of deubiquitinating enzymes have recently been isolated and characterized, their substrates and biological functions have been illustrated. Identified from yeast to human, deubiquitinating (DUB) enzymes are classified into the ubiquitin C-terminal hydrolase (UCH), the ubiquitin-specific processing proteases (UBP or USP), Jab1/Pad1/MPN domain containing metallo-enzymes (JAMM), Otu domain ubiquitin-aldehyde binding proteins (OTU), and Ataxin-3/Josephin domain containing proteins (Ataxin-3/Josephin). Several members of a novel DUB subfamily induced by cytokines in murine lymphocytes have recently been identified. In addition, human DUB enzyme DUB-3, highly homologous to USP17 and induced by cytokines interleukin (IL)-4 and IL-6, has been recently isolated and showed that it has significant homology to the known murine DUB subfamily members. Interestingly, both murine DUB and human USP17 subfamily members are localized and clustered on murine chromosome 7 and on human chromosomes 4 and 8, respectively. This review introduces the reader to provide a great understanding of cytokine-inducible DUB enzymes in both mouse and human and new insights into DUB subfamily members.

The proteasome is a multicatalytic protease complex that degrades most endogenous proteins including misfolded or damaged proteins to ensure normal cellular function. The ubiquitin-proteasome degradation pathway plays an essential role in multiple cellular processes, including cell cycle progression, proliferation, apoptosis and angiogenesis. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that a proteasome inhibitor could be used as a novel anticancer drug. Indeed, this idea has been supported by the encouraging results of the clinical trials using the proteasome inhibitor Bortezomib (Velcade, PS-341), a drug approved by the US Food and Drug Administration (FDA). Though successful in improving clinical outcomes when Bortezomib was used in hematological malignancies, relapse often occurs in those patients who responded initially. Recently, several second-generation of proteasome inhibitors (including carfilzomib, marizomib, and MLN9708) have been applied in clinics. Furthermore, several natural compounds, including the microbial metabolite lactacystin, green tea polyphenols, and traditional medicinal triterpenes, have been shown to be potent proteasome inhibitors. These findings suggest the potential use of natural proteasome inhibitors as not only chemopreventive and chemotherapeutic agents, but also tumorsensitizers to conventional radiotherapy and chemotherapy. In this eBook chapter, we will summarize the structure and biological activities of the proteasome and several natural compounds with proteasome-inhibitory activity, and will discuss the potential use of these compounds for the prevention and treatment of human cancers.

The consideration of protein molecules as graphs whose nodes and edges are respectively the aminoacid residues and the non covalent contacts between them permits to develop very effective models of protein behavior. The network paradigm not only allows for a drastic reduction of the amount of information needed to represent a protein 3D structure, but provides a set of quantitative descriptors derived from mathematical graph theory endowed with crucial information as for protein biological role. Here we will briefly comment about the relevance of graph descriptors in registering the relevant features of the molecular dynamics of solvation process and sketch the possibility to consider contact map as the ‘macromolecular’ analogue of small organic molecules structural formula.

Pure organic solvents or their mixtures with water are commonly used as artificial media for biotechnological application and for fundamental research on protein stability and folding mechanism. The molecular interactions between these environments and biological molecules are complex and variegated, and only recently we started to shade a light on their nature. Molecular modeling methods are effective tools to address the atomistic detail of these processes. In particular, Molecular Dynamics simulation is one of the most powerful and versatile tools to investigate the solvation of complex molecules. In the last few years, the number of publications on peptide and protein simulations in non-natural environments has rapidly increased. These studies are providing important information to shed light on the nature of nonaqueous solvent effects. In this chapter, the achievements and the future prospects in this field of computational biochemistry are reviewed by summarizing the most important theoretical results published in the last 15 years.

The Helmholtz free energy, F and the entropy, S are related thermodynamic quantities with a special importance in structural biology. We describe the difficulties in calculating these quantities and review recent methodological developments, concentrating on the calculation of the absolute free energy of protein-ligand binding. A protein typically resides in a single microstate (a limited region in space, such as the α-helical region of a peptide), but sometimes can interconvert among several microstates at thermodynamic equilibrium. Since this flexibility is essential for protein function we discuss the problems involved in the definition, simulation, and free energy calculation of microstates. While the review is broad, a special emphasis is given to methods for calculating the absolute F (and S), where our HSMD method is discussed in some detail.

In the last years there has been an increasing amount of experimental evidence pointing out that a large number of proteins are either fully or partially disordered. Intrinsically disordered proteins are ubiquitous proteins that fulfil biological functions while lacking highly populated and uniform secondary and tertiary structure under physiological conditions. Despite the frequent occurrence of structural disorder, disordered regions are still poorly detected. Recognition of disordered regions in a protein is instrumental for reducing spurious sequence similarity during sequence comparisons between disordered regions and ordered ones, and for delineating boundaries of protein domains amenable to crystallization. As none of available methods for prediction of protein disorder can be taken as fully reliable on its own, we present a brief overview of current methods and highlight their subjacent philosophy. We show a few practical examples of how they can be combined to avoid respective pitfalls and achieve more reliable predictions. We also describe currently available methods for the identification of regions involved in induced folding and provide a few practical examples in which the accuracy of predictions was experimentally confirmed.

Large volumes of protein sequence and structure data acquired by proteomic studies led to the development of computational bioinformatic techniques that made possible the functional annotation and structural characterization of proteins based on their primary structure. It has become evident from genome-wide analyses that many proteins in eukaryotic cells are either completely disordered or contain long unstructured regions that are crucial for their biological functions. The content of disorder increases with evolution indicating a possibly important role of disorder in the regulation of cellular systems. Transcription factors are no exception and several proteins of this class have recently been characterized as premolten/molten globules. Yet, mammalian cells rely on these proteins to control expression of their 30,000 or so genes. Basic region: leucine zipper (bZIP) DNA-binding proteins constitute a major class of eukaryotic transcriptional regulators. This review discusses how conformational flexibility “built” into the amino acid sequence allows bZIP proteins to interact with a large number of diverse molecular partners and to accomplish their manifold cellular tasks in a strictly regulated and coordinated manner.

α1-Acid Glycoprotein (AGP) is a glycoprotein which increases in concentration in the plasma two to five folds in humans (but also ten to twenty folds in some animals) as a consequence of the systemic response to the inflammation, the so called acute phase reaction. The protein exposes on its surface five N-linked glycans, which exhibit a high degree of subtle structural variations resulting in the expression in blood of several isoforms which have identical amino acid sequence, but different glycosylation patterns. The relative occurrence of these glycoforms are strictly dependent on the pathophysiological status, that is determined by cytokines and hormones produced during inflammation.

Both cytokines and hormones induce changes in the post-translational modification of the protein, and these modifications are independent on the rate of synthesis of the AGP. This review will be subdivided in two parts. The first part will describe the structure of AGP (amino acid sequence and glycan pattern) and the several biological functions identified so far for this protein. The second part will be devoted to the posttranslational modifications of the glycan pattern of AGP due to pathological states, since one of the most interesting features of AGP is that its oligosaccharides microheterogeneity is profoundly affected by pathologic conditions and different glycoforms can appear in plasma during systemic inflammation or diseases.

The impact of different AGP glycoforms on both its transport and anti-inflammatory features and how the modifications of the glycan pattern can be utilized in clinical biochemistry will also be discussed.

Evidence has accumulated that carbohydrate-peptide epitopes do play a role in classical MHC-mediated immune responses. T-cell recognition of O-glycosylated peptides is potentially of high biomedical significance, because it can mediate the immune protection against microorganisms, and in particular the vaccination in anti-tumor therapies.

The epithelial type 1 transmembrane mucin MUC1 is established as a marker for monitoring recurrence of breast cancer and is a promising target for immunotherapeutic strategies to treat cancer by active specific immunization. Natural human immune responses to the tumor-associated glycoforms of the mucin indicate that antibody reactivities are more directed to glycopeptide than to non-glycosylated peptide epitopes. To overcome the weak immunogenicity of the tumor-associated glycoform of MUC1 in experimental immunization, attempts were made to get insight into the molecular requirements for effective antigen processing and to identify class I and II processing permissive glycosylation sites. Evidence based on work with CD4+ T-hybridomas confirms that O-glycopeptide products of the immunoproteasomal or endosomal processing machineries can be effectively presented to T-cells and that glycans can form integral parts of the TCR defined epitopes. Immunization strategies in human MUC1 transgenic mice have demonstrated that different from nonglycosylated epitopes the glycoforms are recognized as foreign by the immune system and can effectively break immunotolerance. Based on these findings superior vaccines have been designed and successfully applied in transgenic mice that have a multi-epitope composition in common by comprising self-adjuvanting, APC-targeting and antigen-specific epitopes.

In recent years, a strong emphasis has been given in deciphering the function of genes unraveled by the completion of several genome sequencing projects. In plants, functional genomics has been massively used in order to search for gene products of agronomic relevance. As far as root-pathogen interactions are concerned, several genes are recognized to provide tolerance/resistance against potential invaders. Root proteins have also been increasingly identified, especially by using recent gel-free proteomic techniques. However, root proteomic analysis is still a challenge, since the major drawbacks, such as low proteins/volume content and high concentration of interfering substances (pigments and phenolic compounds, among others) have not been overcome. This scenario is further hindered by the overall low intrinsic amounts of root tissues in many of the important target plants. The proteome analysis of plant-pathogen interactions provides important information about the global proteins expressed in roots in response to biotic stresses. Several pathogenic proteins superimpose the plant proteome and can be identified and used as targets for the control of viruses, bacteria, fungi and nematode pathogens. The present review focuses on advances in different proteomic strategies dedicated to the challenging analysis of plant defense proteins expressed during bacteria-, fungi- and nematode-root interactions. Recent developments, limitations of the current techniques, and technological perspectives for root proteomics aiming at the identification of resistance-related proteins are discussed.

The myelin basic protein (MBP) family comprises a variety of developmentally-regulated members arising from different transcription start sites, differential splicing, and post-translational modifications. The “classic” isoforms of MBP include the 18.5-kDa form, which predominates in adult human myelin and facilitates compaction of the mature myelin sheath in the central nervous system, thereby maintaining its structural integrity. In addition to membrane-association, the 18.5-kDa and all other classic isoforms are able to interact with a multitude of proteins, including Ca²⁺-calmodulin, actin, tubulin, and SH3-domain-containing proteins, and thus may be signalling linkers during myelin development and remodelling. All proteins in this family are intrinsically disordered, creating a large effective surface to facilitate multiple protein associations, and are post-translationally modified to various degrees by methylation, phosphorylation, and deimination. We have used spectroscopic (fluorescence, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance) and computational (molecular dynamics) approaches to study MBP’s conformational adaptability. A highly-conserved central domain consists of an amphipathic α-helix that associates with a phospholipid membrane. In multiple sclerosis, this segment represents a primary immunodominant epitope. This α-helical structure is adjacent to a proline-rich region that contains a classic SH3-ligand along with two threonyl MAP-kinase phosphorylation sites, and forms a poly-proline type II (PPII) structure. This α-helical segment of the protein is thus essential to proper positioning of the PPII protein-interaction motif, with the local conformation and accessibility being modulated by MAP-kinases, and may represent an important molecular switch. Aberrant post-translational or other modifications in this segment of the protein may participate in the onset and pathogenesis of the human demyelinating disease multiple sclerosis.

After over a century of extensive research, hemoglobin has become the prototype of allosteric and cooperative proteins. Its molecular structure, known in great detail, has allowed the design of hundreds of site directed mutations, aimed at interfering with its function, and thus at testing our hypotheses on the molecular mechanisms of allostery. The wealth of information thus obtained is difficult to read except for specialists, not only because it makes use of many different technical approaches, but also because of its intrinsically patchy nature. Moreover, several researchers have tried to assign specific roles to segments of the polypeptide chains, rather than to single residues, and have tested their hypotheses by multiple point mutations or by complete replacement with the homologous segment from a different hemoglobin to produce chimeric macromolecules. This approach is in great need of a revision since putative functionally relevant segments partially overlap. This review briefly describes the structure and function of hemoglobin, and analyzes the effect of point mutations, multiple mutations and segment replacement, with special attention to possible biotechnological applications, ranging from pharmacology (Hb solutions as resuscitating fluids and sources of the protein found in hemoglobinopathies for biochemical studies) to bioreactors. Occasional reference is made to site directed mutants of myoglobin, whenever this helps clarifying perplexing results obtained on hemoglobin.

Proteins are endowed with the “Lego property”, i.e., the capability of steric fitting with other proteins to form high molecular weight complexes with emergent functions. These interactions may occur both as horizontal molecular networks at the plasma membrane level and as vertical molecular networks, i.e., towards the extra- and/or intracellular side of the cell. The present paper broadens this view by proposing the existence of three dimensional molecular networks, mainly made by proteins and carbohydrates, which might interact with each other at boundaries of compartments such as plasma membranes to form a “global molecular network” (GMN) that pervades the intraas well as the extra-cellular environment of the entire central nervous system. The GMN is a potentially plastic structure regulated through several means. For example, its extracellular part is under the remodeling action of the matrix metalloproteinases.

The proposal of a GMN has physiological and pathological implications.

In primis, classical synaptic transmission, gap junctions and volume transmission signals by modulating GMN could importantly contribute to the “binding phenomenon”, i.e., the phase synchronization of firing rates in far-located neuronal cortical groups.

Secondly, alterations in protein conformation could alter the GMN organization and hence the neuronal network morphology and function. This could lead to the formation of abnormal protein aggregates such as amyloid plaques and neurofibrillary tangles, which, in turn, might affect the GMN function and/or the reciprocal interactions between its parts especially at the boundaries between compartments.

Glycoconjugates comprise a variety of structures, include glycoproteins and glycolipids and are found on the surfaces of animal and plant cells, as well as on the surface of microorganisms. Determination of the structure and the distribution of glycoconjugates on cell surfaces are important for the understanding their biological function. Lectins are useful to investigate protein-carbohydrate interactions, because they have specificity for defined carbohydrate structure. They have been implicated in cell-to-cell recognition and signaling, blood group typing, in immune recognition process, and various other biological processes, such as viral, bacterial, mycoplasmal and parasitic infections, fertilization, cancer metastasis, growth and differentiation. Once thought to be confined to plant seeds, lectins are now recognized as ubiquitous in virtually all living systems, ranging from viruses and bacteria to animals. Plant lectins provide a rich source of carbohydrate-recognizing protein reagents for glycobiologists and biotechnologists. Biotechnology offers the therapeutic use of lectin against certain life threatening diseases such as human immunodeficiency virus and cancer. This review presents a comprehensive summary of research efforts that focus on the actual and potential uses and advantages of using lectins to target glycoproteins and also glycoproteins to target lectins.