Conformational Aspects of Biological Activity of Functional Fragments of the p21ras Oncoproteins: 4. Address Fragments of the Receptor Oncoproteins

Decapeptide fragments of the src, fgr, fps, and yes oncoproteins were studied by theoretical conformational analysis, and the arrangement of ionized residues in these fragments was found to be complementary to the binding site of p21. The results demonstrated a similarity in conformational properties of these peptides and their structural complementarity to the address fragment of p21. On the basis of this computation, a model of interaction of the p21ras family of oncoproteins with their cellular receptors was suggested.     

Conformational aspects of biological activity of functional fragments of the p21ras oncoproteins. 3. Fragment 34-46 of the native oncoprotein and the decapeptide corresponding to its 35-44 sequence

Theoretical conformational analysis was used to study the spatial structure of a putative binding site responsible for binding of p21 oncoproteins to other oncoproteins. Conformational properties of the isolated address sequence localized in fragment 34-46 of native p21 and of the decapeptide molecule corresponding to the 35-44 sequence in the primary structure of oncoproteins of this family were revealed. Our calculations demonstrated a similarity between the spatial structures of the peptides, which confirms the hypothesis on the identity of their biological functions.     

A peptide inhibitor of the binding site of oncoproteins of the p21ras family

The method of theoretical conformational analysis was used to study the inverse structural problem to determine the amino acid sequence of the peptide molecule capable of inhibiting the site of binding of p21 to cell receptors. At the first stage of the computational experiment, the spatial structure and the conformational possibilities of the binding sites of protein p21 and its cellular receptors were determined. Then the three-dimensional structures of several peptides containing the Arg-Ala-Ala-Glu-Asp site were studied. By varying the number of alanine residues in the adjacent regions of the molecule, the sequence H-Asp1-Ala2-Ala3-Ala4-Arg5-Ala6-Ala7-Glu8-Asp9-Ala10-Ala11--Lys12-QH was chosen, which most adequately simulates the conformational properties of the address fragments of oncoprotein receptors. The peptide-molecule having this primary structure is capable of forming a complex with p21, i.e., blocking the binding site of the oncoprotein by preventing the signal transduction from the oncoprotein to the cell, thereby breaking the cycle of the carcinogenic process.     

Conformational Aspects of Biological Activity of Functional Fragments of the p21ras Oncoproteins: 3. Fragment 34–46 of the Native Oncoprotein and the Decapeptide Corresponding to Its 35–44 Sequence

Theoretical conformational analysis was used to study the spatial structure of a putative binding site responsible for binding of p21 oncoproteins to other oncoproteins. Conformational properties of the isolated address sequence localized in fragment 34–46 of native p21 and of the decapeptide molecule corresponding to the 35–44 sequence in the primary structure of oncoproteins of this family were revealed. Our calculations demonstrated a similarity between spatial structures of the peptides, which confirms the hypothesis on the identity of their biological functions.     

The p21(WAF1) cyclin-kinase inhibitor: in vivo interaction with E1A adenoviral Ad2 and Ad12 oncoproteins

The capability of adenoviral oncoproteins E1A Ad2 and Ad12 to form complexes in vivo with cyclin-kinase inhibitor p21Waf1 has been analysed. The published data confirming direct interaction between E1A and p21Waf1 are insufficient. In the present work, a yeast two-hybrid SRS system was used to investigate the binding of different fragments of E1A Ad2 and Ad12 polypeptides with p21Waf1. We have shown that the full length product of 12S mRNA E1A Ad2 interacts weekly with p21Waf1, whereas the protein corresponding to 13S mRNA E1A Ad12 does not bind to cyclin-kinase inhibitor protein. Moreover, fragments 1-80 (Ad2), 1-29 (Ad12), 1-79 (Ad12), and 105-194 (Ad12) were able to interact with p21Waf1 to some extent. The difference between interacting regions of adenoviral proteins E1A Ad2/5 and Ad12 gives a new information about the mechanism of p21Waf1 functional inactivation and different transforming activity of Ad2/5 and Ad12.     

Conformational aspects of the biological effect of functional fragments of the p21ras oncoprotein family. 2. Fragment 1-16, various sequences]

The conformational analysis data on active ([Val12-Gly13], [Asp12-Gly13] and [Gly12-Asp13]) and passive ([Gly12-Gly13] and [Pro12-Gly13]) modifications of the p21ras family oncoproteins are presented. The activating amino acid substitutions are shown to be accompanied by essential changes in the secondary structure, resulted in the 9-16 fragment spiralization. The spatial structure of the 1-9 fragment does not vary for all the predominant forms of the active and passive analogues. The results of the conformational analysis have been used for studying the structural-functional relationships.     

Sequence alignment approach to pick up conformationally similar protein fragments.

Crystal structure data of globular proteins were used to prepare (phi, psi) probability maps of 20 proteinous amino acids. These maps were compared grid-wise with each other and a conformational similarity index was calculated for each pair of amino acids. A weight matrix, called Conformational Similarity Weight (CSW) matrix, was prepared using the conformational similarity index. This weight matrix was used to align sequences of 21 pairs of proteins whose crystal structures are known. The aligned regions with more than seven contiguous amino acids were further analysed by plotting average weight (W) values of overlapping hepatapeptides in these regions and carrying out curve fitting by Fourier series having TEN harmonics. The protein fragments corresponding to the half-linewidth of peaks were predicted as fragments having similar conformation in the protein pair under consideration. Such an approach allows us to pick up conformationally similar protein fragments with more than 67% accuracy.     

Understanding the targeting of the RB family proteins by viral oncoproteins to defeat their oncogenic machinery

The retinoblastoma (RB) family consists of three genes, RB1, RBL1, and RBL2, that code for the pRb, p107, and pRb2/p130 proteins, respectively. All these factors have pivotal roles in controlling fundamental cellular mechanisms such as cell cycle, differentiation and apoptosis. The founder and the most investigated RB family protein is pRb, which is considered to be the paradigm of tumor suppressors. However, p107 and pRb2/p130 clearly display a high degree of structural and functional homology with pRb. Interestingly, these factors were first identified as physical targets of the Adenovirus E1A oncoprotein. Indeed, RB family proteins are the most important and widely investigated targets of small DNA virus oncoproteins, such as Adenovirus E1A, human papillomavirus E7 and Simian virus 40 large T antigen. By interacting with pRb and with other RB family members, these oncoproteins neutralize their growth suppressive properties, thus stimulating proliferation of the infected cells, de‐differentiation, and resistance to apoptosis. All these acquired features strongly favor the rise and selection of immortalized and mutation‐prone cells, leading to a higher propensity in undergoing transformation. Our present work aims to illustrate and delve into these protein–protein interactions. Considering that these viral oncoproteins are dispensable for normal cellular functions, they can create “oncogene addiction” in the infected/transformed cells. This makes the possibility to dismantle these interactions extremely attractive, thus promoting the development of highly specific smart molecules capable of targeting only the infected/transformed cells that express these viral factors. J. Cell. Physiol. 228: 285–291, 2013. © 2012 Wiley Periodicals, Inc.     

Regulation of nuclear receptor activities by two human papillomavirus type 18 oncoproteins,E6 and E7

The human papillomavirus (HPV) E6 and E7 oncoproteins are two major proteins that remain expressing in HPV-associated human cancers. The high-risk HPVs synthesize E6 and E7 oncoproteins to alter the function of cellular regulatory proteins, such as p53 and retinoblastoma gene product, respectively. In this study, we demonstrated that HPV-18 E6 and E7 proteins were able to directly interact with some nuclear receptors (NRs), such as thyroid receptor, androgen receptor, and estrogen receptor (ER), whether or not appropriate hormones were present. The functional roles of these two oncoproteins in NRs depended on the cell type (including ligand), promoter context, and NR type. These two oncoproteins regulated ER functions through ER's AF-1, AF-2, or both. Hence, our results provide new insights into the mechanisms controlling the proliferation and immortalization of HPV infected cells by these two oncoproteins mediating through their regulatory functions in NR systems.     

Conformational analysis of tachykinins. I. N-terminal fragments of substance P, physalemin, hylambatin, and uperolein]

Theoretical conformational analysis of N-terminal fragments of the title peptides has been carried out using the potential energy calculations. The number of conformational states for each fragment is very limited, and they are easily interconverted. Since these fragments cannot form alpha-helises, it is unlikely that upon binding of tachikinins to their receptors, their N-terminal fragments could overcome the hydrophobic barrier of the cell membrane's lipid belayer.     

Conformational aspects of the biological action of functional fragments of the p21ras oncoprotein family. 1. Fragments 1-11 and 9-16 of the polypeptide chain]

Using theoretical conformational analysis, spatial structures of the N-terminal undecapeptide, common to all p21 modifications, and of the 9-16 fragments of the protein's active and passive analogues have been investigated. The data obtained reveal an essential differences between the predominant backbone forms of the active and passive modifications of the oncoprotein.     

SV40 early region oncoproteins and human cell transformation

We now understand neoplastic transformation to be the consequence of multiple acquired genetic alterations. The combination of these acquired changes confer the various phenotypes that constitute the clinical features of cancer. Although only rare human cancers derive from a viral etiology, the study of DNA tumor viruses that transform rodent and human cells has led to a greater understanding of the molecular events that program the malignant state. In particular, investigation of the viral oncoproteins specified by the Simian Virus 40 Early Region (SV40 ER) has revealed critical host cell pathways, whose perturbation play an essential role in the experimental transformation of mammalian cells. Recent work has re-investigated the roles of two SV40 ER oncoproteins, the large T antigen (LT) and the small t antigen (ST), in human cell transformation. Co-expression of these two oncoproteins, together with the telomerase catalytic subunit, hTERT, and an oncogenic version of the H-Ras oncoprotein, suffices to transform human cells. LT inactivates two key tumor suppressor pathways by binding to the retinoblastoma protein (pRB) and p53. The ability of ST to transform human cells requires interactions with PP2A, an abundant family of serine-threonine phosphatases. Here we review recent developments in our understanding of how these two viral oncoproteins facilitate human cell transformation.     

The effect of amino acid substitutions on the 3-dimensional structure of fragment 1--16 of the oncoprotein p21 ras

Using the method of theoretical conformational analysis, spatial structure of fragment 1-16 of active [( Val12-Gly13], [Asp12-Gly13], [Gly12-Asp13]) and passive [( Gly12-Gly13] and [Pro12-Gly13]) modifications of oncoproteins family p21 ras have been investigated. The activation of these proteins has been shown to be accompanied by reorganization of three-dimensional structure of the polypeptide chain.     

Two dimensional single-strand conformation polymorphism analysis: a useful tool for the detection of mutations in long DNA fragments.

A new two-dimensional gel system for the analysis of single strand conformational polymorphisms has been developed to identify point mutations, deletions and insertions in long DNA fragments (e.g. 2.7 kb) generated by the polymerase chain reaction. In this procedure, such DNA fragments are first restricted with frequent-cutter enzymes. The resulting small fragments are then separated in the first dimension according to their size by electrophoresis under denaturing conditions; these single stranded DNA fragments are subsequently fractionated in the second dimension by electrophoresis on a non denaturing slab gel based on their fold-back conformation which is completely sequence-dependent. The method was tested on three previously characterized pH 4.5 resistant mutants of HRV14 and was then used to determine changes in three further mutants.     

Mechanism of action of aspartic proteinases. V. Conformational characteristics of fragments of substrate-binding sites in rhizopuspepsin and HIV-1 proteinase

The conformational states of side chains of catalytic Asp residues in active sites of HIV-1 protease and rhizopuspepsin in the potential field of free enzymes were studied by using theoretical conformational analysis. Structural factors that stabilize the conformation of these residues in free enzymes were revealed. Methods of molecular mechanics were used to estimate the stabilization energy of the Met46-Phe53 labile fragments of HIV-1 protease in the potential field of their nearest surrounding amino acid residues for the conformations characteristic of the free protein and similar to that of the protein in enzyme-inhibitor complexes. In solution, the conformational state of the fragments of the free enzyme was concluded to be similar to that observed in the enzyme complex with the ligand and different from that determined by X-ray diffraction analysis. This difference was ascribed to the effect of crystal packing.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Peptide fragments as models to study the structure of a G-protein coupled receptor: the alpha-factor receptor of Saccharomyces cerevisiae

The alpha-factor tridecapeptide initiates mating in Saccharomyces cerevisiae upon interaction with Ste2p, its cognate G-protein coupled receptor (GPCR). This interaction is being used as a paradigm for understanding the structure and mechanism of activation of GPCRs by medium-sized peptides. In this article, the use of fragments of Ste2p to study its structure is reviewed. Methods of synthesis of peptides corresponding to both extramembranous and transmembrane domains of Ste2p are evaluated and problems that are encountered during synthesis and purification are described. The results from conformational analyses of the peptide fragments using fluorescence spectroscopy, CD, infrared spectroscopy, and NMR spectroscopy in organic-aqueous mixtures and in the presence of detergent micelles and lipid bilayers are critically reviewed. The data obtained to date provide biophysical evidence for the structure of different domains of Ste2p and indicate that peptides corresponding to these domains have unique biophysical tendencies. The studies carried out on Ste2p fragments indicate that valuable information concerning the structure of the intact receptor can be obtained by studying peptide fragments corresponding to domains of these polytopic integral membrane proteins.     

Mechanisms of tumor suppressor protein inactivation by the human papillomavirus E6 and E7 oncoproteins

There are now several examples where experimental and epidemiologic data have implied a causative role for viruses in human cancer. Human papillomavirus (HPV) DNA is found in approximately 90% of cervical cancers. Only a subset of the HPV types that infect the anogential tissues, however, are associated with cancer. Interestingly, only the cloned DNA of this subset is capable of immortalizing human primary genital keratinocytes in culture. The oncoproteins of the HPVs are encoded by the E6 and E7 genes. Analogous to the oncoproteins of certain other DNA tumor viruses, the E6 and E7 proteins have been shown to functionally inactivate the tumor suppressor proteins p53 and pRB, respectively. We will review what is known of the mechanisms by which the E6 and E7 proteins inactivate these tumor suppressors and the evidence that these activities are related to the transforming capabilities of the HPVs associated with cancer.     

Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex.

Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett., 439, 197-202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem. 267, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.