Structured proteins and proteins with internal disorder

The point of view that a uniquely folded protein tertiary structure is required for the protein functioning has been prevailing in the literature quite recently. However of lately it has been found that many proteins in a cell have no such structure in an isolated state, though they have a well-defined function in physiological conditions. These proteins were named as proteins with natural or internal disorder. The portion of disordered regions in such proteins may vary from a sequence of several amino acids to a completely disordered sequence containing from tens to hundreds of amino acids. The main difference of these proteins from the structured (globular) ones is that they have no unique tertiary structure in an isolated state and acquire it after interaction with their partners. Their conformation in such a complex depends on the interacting partner and not only on their own amino acid sequence, which is specific for structured (globular) proteins. The problem of structural and functional relations in the structured proteins and proteins with internal disorder is discussed in this review. The complexity of the problem and its potential solutions are illustrated by the example of elongation factors EFlA.     

Structured proteins and proteins with intrinsic disorder

Until recently, the point of view that the unique tertiary structure is necessary for protein function has prevailed. However, recent data have demonstrated that many cell proteins do not possess such structure in isolation, although displaying a distinct function under physiological conditions. These proteins were named the naturally, or intrinsically, disordered proteins. The fraction of intrinsically disordered regions in such proteins may vary from several amino acid residues to a completely unordered sequence of several tens or even several hundreds of residues. The main distinction of these proteins from structured (globular) proteins is that they have no unique tertiary structure in isolation and acquire it only upon interaction with their partners. The conformation of these proteins in a complex is determined not only by their own amino acid sequence (as is typical of structured, or globular, proteins) but also by the interacting partner. This review discusses the structure-function relationships in structured and intrinsically disordered proteins. The intricateness of this problem and the possible ways to solve it are illustrated by the example of the EF1A elongation factor family.     

Membrane interactions of G proteins and other related proteins

Guanine nucleotide-binding proteins, G proteins, propagate incoming messages from receptors to effector proteins. They switch from an inactive to active state by exchanging a GDP molecule for GTP, and they return to the inactive form by hydrolyzing GTP to GDP. Small monomeric G proteins, such as Ras, are involved in controlling cell proliferation, differentiation and apoptosis, and they interact with membranes through isoprenyl moieties, fatty acyl moieties, and electrostatic interactions. This protein-lipid binding facilitates productive encounters of Ras and Raf proteins in defined membrane regions, so that signals can subsequently proceed through MEK and ERK kinases, which constitute the canonical MAP kinase signaling cassette. On the other hand, heterotrimeric G proteins undergo co/post-translational modifications in the alpha (myristic and/or palmitic acid) and the gamma (farnesol or geranylgeraniol) subunits. These modifications not only assist the G protein to localize to the membrane but they also help distribute the heterotrimer (Galphabetagamma) and the subunits generated upon activation (Galpha and Gbetagamma) to appropriate membrane microdomains. These proteins transduce messages from ubiquitous serpentine receptors, which control important functions such as taste, vision, blood pressure, body weight, cell proliferation, mood, etc. Moreover, the exchange of GDP by GTP is triggered by nucleotide exchange factors. Membrane receptors that activate G proteins can be considered as such, but other cytosolic, membranal or amphitropic proteins can accelerate the rate of G protein exchange or even activate this process in the absence of receptor-mediated activation. These and other protein-protein interactions of G proteins with other signaling proteins are regulated by their lipid preferences. Thus, G protein-lipid interactions control the features of messages and cell physiology.     

Membrane proteins and membrane proteomics

Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins.     

Glucocorticoids and acute phase proteins

Glucocorticoids as well as acute phase proteins participate in non-specific host defence as well as in restoring host integrity after injury. Plasma levels of both compounds augment during the inflammatory reaction. However, glucocorticoids also have physiological effects that share similar molecular mechanisms with the family of steroids. During the inflammatory reaction, and for participating in host defense, glucocorticoids, together with augmented cytokines, use new signalling pathways. In doing so, they participate in the positive or negative control of inflammatory mediator synthesis. For example, they induce the synthesis of acute phase proteins in synergy with interleukin 6, interleukin 1 and TNF alpha.     

TRP proteins and cancer

Cancer is the second most common cause of death in western countries. It is therefore of fundamental importance to improve the treatment of patients with malignant tumors. This goal can only be achieved if we get closer insight in the various mechanisms leading to tumor formation. Significant progress in the understanding of carcinogenesis has been made during the last couple of years. Ion channels contribute to the regulation of cell proliferation which has initially been shown for K+ channels. Meanwhile, other ion channels such as Cl-, Na+ and Ca2+ channels seem to influence cellular function like growth, migration and invasion. In addition, cation channels of the transient receptor potential (TRP) superfamily are implicated in cancer formation. Most recent data concerning TRP vanilloid (TRPV) type 6, TRP melastatin (TRPM) type 1 and 8 channels and their relevance for common human cancer types will be highlighted in this review. Furthermore, TRP channel structure and function will be discussed in the light of their possible importance as prognostic markers and targets for drug discovery.     

RGS proteins have a signalling complex: interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins

The intracellular regulator of G protein signalling (RGS) proteins were first identified as GTPase activating proteins (GAPs) for heterotrimeric G proteins, however, it was later found that they can also regulate G protein-effector interactions in other ways that are still not well understood. There is increasing evidence that some of the effects of RGS proteins occur due to their ability to interact with multiprotein signalling complexes. In this review, we will discuss recent evidence that supports the idea that RGS proteins can bind to proteins other than Galpha, such as G protein coupled receptors (GPCRs, e.g. muscarinic, dopaminergic, adrenergic, angiotensin, interleukin and opioid receptors) and effectors (e.g. adenylyl cyclase, GIRK channels, PDEgamma, PLC-beta and Ca(2+) channels). Furthermore, we will investigate novel RGS binding partners (e.g. GIPC, spinophilin, 14-3-3) that underlie the formation of signalling scaffolds or govern RGS protein availability and/or activity.     

Calcium-binding proteins and secretion

The Ca ion plays a central role in the control of the regulated pathway of exocytotic secretion in eukaryote cells. Most secretagogues either directly or indirectly raise cytosolic free Ca levels which in turn affects granule biogenesis, contractile events, gel/sol transition in intracellular matrix and membrane fusion events occurring at exocytosis. Many of these responses are mediated by Ca-binding proteins among which calmodulin and protein kinase C have received prominent attention. Studies of the nature and inter-relationship of proteins which undergo Ca-dependent association with intracellular membranes in secretory tissue reveal that there may be further Ca-binding proteins in these cells which act as intracellular transducers of the Ca signal during secretion.     

Micropatterned agarose gels for stamping arrays of proteins and gradients of proteins

We describe a method for repetitive and rapid formation of planar microarrays and gradients of proteins using patterned agarose stamps. It demonstrates: (i) micropatterning of agarose gels with feature sizes as small as 2 microm; (ii) inking of posts (diameter 50-1000 microm) on patterned agarose stamps with one or multiple (here, eight) proteins and repetitive stamping of patterns (>100 times in the case of one protein) and arrays (20 times in the case of eight proteins) without the need for intermediate re-inking; (iii) transferring spots of proteins with good homogeneity in surface coverage to glass slides; (iv) applying this technique to surface-based immunoassays; (v) stamping that requires only sub-nanomolar amounts of protein (typically approximately 3 microg in approximately 0.6 microL of solution); (vi) stamping without the need for drying of the proteins, as opposed to stamping with stamps made of poly(dimethylsiloxane); and (vii) patterning gradients of proteins by allowing two proteins to diffuse toward each other in an agarose stamp, followed by printing the protein gradients onto a surface.     

Quantum dots and prion proteins

A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels.     

Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activities

Both recombinant full-length mouse prion protein expressed in Escherichia coli and native prion protein (PrPsc) from mouse brain exhibited NADH oxidase and protein disulfide-thiol interchange activities similar to those formerly thought to be properties exclusive to the growth-related, cell surface ECTO-NOX proteins. The two activities exhibited the complex 2+3 pattern of oscillations characteristic of ECTO-NOX proteins where the two activities alternate to generate a period length of 24 min. The oscillations were augmented by copper and diminished by addition of the copper chelator bathocuproene. That the activity might be attributable to a contaminating protein was ruled out by experiments where the purified recombinant prion-containing extracts were resolved by SDS-PAGE and the activity was restricted to a single band corresponding to the predicted Mr of the recombinant prion as verified by Western blot analyses.     

Autophagic proteins

Oxygen (O₂), while essential for aerobic life, can also cause metabolic toxicity through the excess generation of reactive oxygen species (ROS). Pathological changes in ROS production can originate through the partial reduction of O₂ during mitochondrial electron transport, as well as from enzymatic sources. This phenomenon, termed the oxygen paradox, has been implicated in aging and disease, and is especially evident in critical care medicine. Whereas high O₂ concentrations are utilized as a life-sustaining therapeutic for respiratory insufficiency, they in turn can cause acute lung injury. Alveolar epithelial cells represent a primary target of hyperoxia-induced lung injury. Recent studies have indicated that epithelial cells exposed to high O₂ concentrations die by apoptosis, or necrosis, and can also exhibit mixed-phenotypes of cell death (aponecrosis). Autophagy, a cellular homeostatic process responsible for the lysosomal turnover of organelles and proteins, has been implicated as a general response to oxidative stress in cells and tissues. This evolutionarily conserved process is finely regulated by a complex interplay of protein factors. During autophagy, senescent organelles and cellular proteins are sequestered in autophagic vacuoles (autophagosomes) and subsequently targeted to the lysosome, where they are degraded by lysosomal hydrolases, and the breakdown products released for reutilization in anabolic pathways. Autophagy has been implicated as a cell survival mechanism during nutrient-deficiency states, and more generally, as a determinant of cell fate. However, the mechanisms by which autophagy and/or autophagic proteins potentially interact with and/or regulate cell death pathways during high oxygen stress, remain only partially understood.     

Vitamin E and transfer proteins

PURPOSE OF REVIEW: Recently, the intracellular transport as well as cellular uptake and excretion of alpha-tocopherol, the major representative of vitamin E, have been elucidated. RECENT FINDINGS: Alpha-tocopherol transfer protein has been identified as the major intracellular transport protein for vitamin E, mediating alpha-tocopherol secretion into the plasma via a non-Golgi-dependent pathway, while other binding proteins seem to play a less important role. New information has accumulated concerning the role of this protein in the transport and supply of vitamin E to tissues such as the central nervous system and the feto-maternal unit. The scavenger receptor class B type I receptor, a membrane-bound protein, is capable of transferring vitamin E into the cell, while the ATP-binding cassette transporter A1 can excrete vitamin E out of the cell. Advances in the area of vitamin E metabolism have shown that alpha-CEHC (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman) and gamma-CEHC (2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman) are formed by a cytochrome p450-mediated process, important for alpha and gamma-tocopherol excretion. SUMMARY: Insights into the regulation of vitamin E transport and metabolism on the cellular level have made enormous advances, showing the complex interplay of influx, trafficking, efflux and metabolism of this crucial antioxidant.     

Nuclear pore proteins and cancer

Nucleocytoplasmic trafficking of macromolecules, a highly specific and tightly regulated process, occurs exclusively through the nuclear pore complex. This immense structure is assembled from approximately 30 proteins, termed nucleoporins. Here we discuss the four nucleoporins that have been linked to cancers, either through elevated expression in tumors (Nup88) or through involvement in chromosomal translocations that encode chimeric fusion proteins (Tpr, Nup98, Nup214). In each case we consider the normal function of the nucleoporin and its translocation partners, as well as what is known about their mechanistic contributions to carcinogenesis, particularly in leukemias. Studies of nucleoporin-linked cancers have revealed novel mechanisms of oncogenesis and in the future, should continue to expand our understanding of cancer biology.     

Growth-regulated proteins and neuronal plasticity

Growth-regulated proteins (GRPs) of the neuron are synthesized during outgrowth and regeneration at an increased rate and enriched in nerve growth cones. Therefore, they can be used to some degree as markers of neurite growth. However, these proteins are not unique to the growing neuron, and their properties are not known sufficiently to assign them a functional and/or causal role in the mechanisms of outgrowth. During synaptogenesis, GRPs decrease in abundance, and growth cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release. However, there is a stage during which growth cone and synaptic properties overlap to some degree. We propose that it is this overlap and its continuation that allow for synaptic plasticity in developing and adult nervous systems. We also propose a hypothesis involving (a) trophic factor(s) that might explain the regulation of synaptic sizes and collateral sprouting. Some GRPs, especially GAP43/B50/pp46/F1, are more prominent in adult brain regions of high plasticity, and they undergo change, such as phosphorylation, during long-term potentiation (LTP). Without precise functional knowledge of GRPs, it is impossible to use changes in such proteins to explain the plasticity mechanism. However, changes in these "growth markers" are likely to be an indication of sprouting activity, which would explain well the various phenomena associated with plasticity and learning in the adult. Thus, plasticity and memory may be viewed as a continuation of the developmental process into adulthood.     

Recombinant avidin and avidin-fusion proteins

Both chicken egg-white avidin and its bacterial relative streptavidin are well known for their extraordinary high affinity with biotin (Kd approximately 10(-15) M). They are widely used as tools in a number of affinity-based separations, in diagnostic assays and in a variety of other applications. These methods have collectively become known as (strept)avidin-biotin technology. Biotin can easily and effectively be attached to different molecules, termed binders and probes, without destroying their biological activity. The exceptional stability of the avidin-biotin complex and the wide range of commercially available reagents explain the popularity of this system. In order by genetic engineering to modify the unwanted properties of avidin and to further expand the existing avidin-biotin technology, production systems for recombinant avidin and avidin-fusion proteins have been established. This review article presents an overview of the current status of these systems. Future trends in the production and applications of recombinant avidin and avidin-fusion proteins are also discussed.