Protein epitope mimetics as anti-infectives

There is growing interest in the design of synthetic molecules that mimic the structures and functions of epitopes found on the surface of peptides and proteins. Epitope mimetics can provide valuable tools to probe complex biological processes, as well as interesting leads for drug and vaccine discovery. One application of epitope mimetics is reviewed here, focusing on mimetics of the cationic antimicrobial peptides that form part of the innate immune response to microbial and viral infection in many organisms. Mimetics of these naturally occurring peptides and proteins may be useful to explore mechanisms of antimicrobial and immunomodulatory action, and as a potential source of new antibiotics to address one of the most pressing current threats to human health.     

Protein sequences as random fractals

The analysis of primary sequences from a protein sequence data base suggests that the sequences can be considered as examples of constrained random fractals. Fractal dimensions of the positional distributions of the 20 residues along the chain have been calculated. These fractal dimensions can be used as indices of intrinsic preferences of various residues.     

Protein and polypeptide hormones as inhibitors

It is becoming increasingly apparent that hormones regulate morphogenetic processes and metabolic reactions through inhibitory mechanisms as well as stimulatory effects. Inhibition by protein and polypeptide hormones and the numerous analogs that have been prepared, was discussed in a recent article (1). The present brief review is concerned with more recent developments in this field, including the role of protein and polypeptide hormones in the control of cell proliferation and differentiation.     

Protein epitope mimetic macrocycles as biopharmaceuticals

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Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25–30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25–30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100–200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage. Received: 21 December 2000 / Accepted: 28 February 2001     

Polymeric Microspheres as Protein Transduction Reagents

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.Many proteomic techniques can be used to build a picture of a protein with unknown function, but eventually the individual protein''s activity must be studied. Traditional transfection of encoding DNA permits intracellular expression, but often at uncontrolled, nonphysiological levels. Moreover, DNA transfection can neither deliver protein–inhibitor complexes nor readily deliver multiple proteins in a single experiment and thus exploit knowledge from proteomic protein–protein interaction analyses. In contrast, a truly generic protein transduction reagent could theoretically address all possibilities. We believe that polymeric microspheres could fulfill this role, and we have recently synthesized and characterized dual-functionalized, bio-compatible microspheres that permit intracellular tracking (1). Herein, we now report the development of those microspheres into a protein transduction reagent that can carry protein stably, deliver it efficiently to cells, release the protein in the cytoplasm, and concurrently permit fluorescent imaging of transduced cells.Phagocytosis of microspheres was first observed over 30 years ago (2). Perhaps more unexpectedly, uptake of polystyrene microspheres has recently been reported in many other, nonphagocytic cell types, some of which are traditionally considered to be resistant to DNA transfection and/or protein transduction. For example, microspheres are taken up readily by primary immune cells (3), embryonic stem cells (4), human neural stem cells (5), differentiating mouse neural stem cells (5), and several nonphagocytic cell lines (3, 6, 7). In all instances, the reported efficiency of cellular uptake is high, with “beadfection” of up to 90% of cells being typical (4, 5, 8). No additional reagents aside from the microspheres themselves are required in order to promote cellular uptake, and critically, no toxicity has been observed in any of the cell types beadfected, including HEK293T and L929 cells 2 days after beadfection (8), E14g2a embryonic stem cells 3 days after beadfection (4), and mouse and human neural stem cells 30 days after beadfection (5). In the latter case, the microspheres did not have any deleterious effect on the differentiation of human neural stem cells 30 days after beadfection (5).The mechanism of microsphere entry is also nontoxic, and compelling evidence has been published recently that polystyrene-based microspheres (from 0.2 μm to as large as 2 μm) enter cells via a non-endocytosis, energy-independent mechanism (8). Although unusual, such a mechanism is consistent with claims for the commercial reagent Chariot™ (9). Interestingly, a non-endocytic, energy-independent mechanism has also been reported for the entry of rhenium cluster/polymer hybrid particles into HeLa cells (10). Failure of the microspheres to be endocytosed, at least via a clathrin-dependent mechanism, is perhaps to be predicted, as their diameter considerably exceeds that of clathrin-coated vesicles (typically 100 nm). Bradley and co-workers (8) propose that the entry mechanism for polystyrene-based microspheres is one of passive diffusion in which the microsphere interacts with the membrane, anchors, and, after membrane reorganization, enters the cell, resulting in direct cytoplasmic localization.For functional analysis following transduction, the avoidance of endocytosis or phagocytosis is particularly relevant, as endocytosed particles are destined for endosomes and then, normally, for the lysosomes. The lowered pH of the endosome and, more seriously, the acidic and hydrolytic environment of the lysosome risk disruption of the protein structure and/or function. In contrast, for vaccine delivery (where liposomes can be employed), such exposure is advantageous because protein breakdown forms an essential part of antigen presentation. The potential for protein breakdown in endosomes is also irrelevant for the delivery of protein/peptide drugs such as insulin (for which microencapsulation has proven effective for long-term controlled drug release (11, 12)), as these drugs typically function in the extracellular environment, often exerting their effects by binding to membrane-bound receptors. Thus, although vehicles such as liposomes and nanoparticles are employed both extensively and successfully as drug and vaccine delivery vectors in vivo (13–16), they are far from ideal for studying the biological effect of a delivered protein in vitro. Colloidal particles are also endocytosed (17), and therefore these delivery vehicles may present similar disadvantages.Traditionally, protein transduction domains such as HIV TAT (18–20) or other cell-penetrating peptides (21–23) are used to deliver proteins to cells. Whereas positively charged peptides such as TAT are thought to enter the cells via macropinocytosis (reviewed in Ref. 24), a recent publication suggests that at least some cell-penetrating peptide/bio-cargo complexes (siRNA) are endocytosed (25). Here, although the cargoes avoid the lysosomes, acidification of the endosome is required for endosomal escape of the delivered cargo, and indeed, acidification appears to be a recurring requirement for endosomal escape of biomolecular cargoes using cell-penetrating peptides (reviewed in Ref. 24). Consequently, cell-penetrating peptides are unlikely to become generic tools for functional protein delivery.In contrast, the recent demonstrations that polystyrene microspheres can carry a variety of molecular cargoes with them into the cytoplasm (4, 5, 7, 26, 27) make them particularly exciting as potential vectors for delivering functional proteins and/or protein complexes. β-Galactosidase retains its activity when delivered via this route (7), confirming the potential of microspheres to act as generic protein-delivery vehicles. However, delivered proteins have to date remained tethered to the microspheres, and thus existing studies are limited to proteins that are active in the cytoplasm and, critically, retain their activity when immobilized on polystyrene. For the broad-based study of protein function, the subsequent release of the delivered protein within the cell is desirable.An ideal technology would deliver any protein to any cell type and release that protein in the cell, where it could undertake its normal activity. Here we report the first example of such a microsphere-based approach. Protein is delivered on microspheres and then released in the cell by the reducing cytoplasmic environment. This release is mediated by a linker that attaches the protein stably and covalently to the microspheres in vitro but intracellularly is cleaved over a period of hours. It has already been shown that microspheres are taken up with high efficiency by a range of cell types and can carry a variety of cargoes. Because the chemistry of the linker described herein is amenable to linkage with any molecule containing a free amine moiety, the technology provides a new generic platform for in vitro, cell-based delivery of individual proteins, protein complexes, protein mixtures, or other amino-functionalized molecules.     

Protein as an edited random copolymer

It is widely believed that the unique primary structure of a given protein is quite necessary for its folding into a certain three-dimensional structure as well as for its functioning and is a result of a directed selection in the course of biological evolution. The present paper provides arguments in favour of an alternative point of view according to which typical three-dimensional structures of globular proteins are characteristic even for random sequences of amino acid residues. Therefore it may be possible that primary structures of proteins are mainly examples of random amino acid sequences slightly edited in the course of biological evolution to impart them some additional (functional) meaning.     

Detecting Protein Complexes in Protein Interaction Networks Modeled as Gene Expression Biclusters

Developing suitable methods for the detection of protein complexes in protein interaction networks continues to be an intriguing area of research. The importance of this objective originates from the fact that protein complexes are key players in most cellular processes. The more complexes we identify, the better we can understand normal as well as abnormal molecular events. Up till now, various computational methods were designed for this purpose. However, despite their notable performance, questions arise regarding potential ways to improve them, in addition to ameliorative guidelines to introduce novel approaches. A close interpretation leads to the assent that the way in which protein interaction networks are initially viewed should be adjusted. These networks are dynamic in reality and it is necessary to consider this fact to enhance the detection of protein complexes. In this paper, we present “DyCluster”, a framework to model the dynamic aspect of protein interaction networks by incorporating gene expression data, through biclustering techniques, prior to applying complex-detection algorithms. The experimental results show that DyCluster leads to higher numbers of correctly-detected complexes with better evaluation scores. The high accuracy achieved by DyCluster in detecting protein complexes is a valid argument in favor of the proposed method. DyCluster is also able to detect biologically meaningful protein groups. The code and datasets used in the study are downloadable from https://github.com/emhanna/DyCluster.     

Protein kinase C as a stress sensor

While there are many reviews which examine the group of proteins known as protein kinase C (PKC), the focus of this article is to examine the cellular roles of two PKCs that are important for stress responses in neurological tissues (PKC gamma and epsilon) and in cardiac tissues (PKC epsilon). These two kinases, in particular, seem to have overlapping functions and interact with an identical target, connexin 43 (Cx43), a gap junction protein which is central to proper control of signals in both tissues. While PKC gamma and PKC epsilon both help protect neural tissue from ischemia, PKC epsilon is the primary PKC isoform responsible for responding to decreased oxygen, or ischemia, in the heart. Both do this through Cx43. It is clear that both PKC gamma and PKC epsilon are necessary for protection from ischemia. However, the importance of these kinases has been inferred from preconditioning experiments which demonstrate that brief periods of hypoxia protect neurological and cardiac tissues from future insults, and that this depends on the activation, translocation, or ability for PKC gamma and/or PKC epsilon to interact with distinct cellular targets, especially Cx43. This review summarizes the recent findings which define the roles of PKC gamma and PKC epsilon in cardiac and neurological functions and their relationships to ischemia/reperfusion injury. In addition, a biochemical comparison of PKC gamma and PKC epsilon and a proposed argument for why both forms are present in neurological tissue while only PKC epsilon is present in heart, are discussed. Finally, the biochemistry of PKCs and future directions for the field are discussed, in light of this new information.     

Protein patterns as codes for tumor identification

Particulate-fraction and soluble-fraction proteins from examples of breast carcinoma, Hodgkin's lymphoma, and colon adenocarcinoma and their corresponding normal tissues were resolved on sodium dodecyl sulfate-polyacrylamide gels. The Coomassie blue-stained protein patterns were quantitated using a scanning densitometer and were reduced to simple three-part numerical codes. The codes were (a) specific for each tissue and (b) in the examples of lymphoma and colon adenocarcinoma, indicated a strong similarity between tumors and their respective normal tissues.     

Protein kinases as targets for anti-parasitic chemotherapy

Parasitic protozoa infecting humans have a staggering impact on public health, especially in the developing world. Furthermore, several protozoan species are major pathogens of domestic animals and have a considerable impact on food production. In many instances, the parasites have developed resistance against available chemotherapeutic agents, making the search for alternative drugs a priority. In line with the current interest in protein kinases inhibitors as potential drugs against a variety of diseases, the possibility that protein kinases may represent targets for novel anti-parasitic agents is being explored. Research into parasite protein kinases has benefited greatly from genome and EST sequencing projects, with the genomes of a few species fully sequenced (notably that of the human malaria parasite Plasmodium falciparum) and several more under way. The overall picture that emerged from research in this area shows that the phylogenetic isolation of parasitic protozoa is reflected by atypical structural and functional properties of many of their protein kinase homologues. Likewise, evidence is emerging, which suggests that the organisation of some otherwise well-conserved signal transduction pathways is divergent in some parasitic species. The differences between protein kinases of a parasite and their homologues in its host cell suggest that specific inhibition of the former can be achieved. The development of anti-parasitic drugs based on protein kinase inhibition is being pursued following two avenues: one consists of screening chemical libraries on recombinant enzymes; several protein kinases from parasitic protozoa are now available for this approach. The second approach relies on the identification of the molecular targets of kinase inhibitors which display anti-parasitic properties. This has led to promising developments in a few instances, in particular regarding PKG as a drug target against Eimeria and Toxoplasma, and purvalanol B, a purine-based CDK inhibitor which appears to affect unexpected targets in several protozoan parasites. The recent resolution of the structure of a Plasmodium protein kinase complexed with small inhibitory molecules opens the way to a rational approach towards the design of anti-parasitic drugs based on kinase inhibition.     

Protein aggregation as a paradigm of aging

The process of physiological decline leading to death of the individual is driven by the deteriorating capacity to withstand extrinsic and intrinsic hazards, resulting in damage accumulation with age. The dynamic changes with time of the network governing the outcome of misfolded proteins, exemplifying as intrinsic hazards, is considered here as a paradigm of aging. The main features of the network, namely, the non-linear increase of damage and the presence of amplifying feedback loops within the system are presented through a survey of the different components of the network and related cellular processes in aging and disease.     

多功能的胞内衔接蛋白syntenin

Syntenin蛋白是在原核生物及真核生物中广泛存在的一类胞内衔接蛋白（adaptor proteins）. Syntenin由N端结构域（N-terminal domain,NTD）、两个串联的PDZ结构域(postsynaptic density protein, disc large and zonula occludens, PDZ)和C端结构域（C-terminal domain,CTD）组成,在生物进化过程中相对保守. Syntenin蛋白的PDZ结构域可与不同膜受体C端的PDZ结合基序（PDZ-binding motif,PBM）特异性结合, PDZ结构域结合受体的多样性导致了syntenin功能的多样性. 本文综述了syntenin蛋白的发现与分布及其结构特征,对syntenin在肿瘤转移、细胞质膜蛋白组装、参与动物免疫等领域的研究成果进行了较为详细的综述,同时介绍了syntenin在参与动物胚胎发育调控、血管生成和轴突生长等方面的研究进展.     

Protein microarrays as tools for functional proteomics

Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays.