[Computer simulation of complex formation between plastocyanine and cytochrome f in thylakoid lumen].

The diffusion of the protein plastocyanine and complex formation between plastocyanine and cytochrome f (a subunit of a cytochrome b6/f complex) in the chloroplast thylakoid lumen has been studied. A 3D computer simulation model of diffusion and binding of plastocyanine and cytochrome f has been constructed, which considers their electrostatic interaction. Based on the experimental data, the parameters of the model for complex formation between plastocyanine and cytochrome f in solution have been estimated. The dependence of the rate of plastocyanine-cytochrome f reaction on the size of the luminal space has been studied. It was shown that the contraction of the luminal space leads to a decrease in the reaction rate, which is in agreement with the experimental data on the inhibition of the reaction under hyperosmotic stress.

Direct simulation of plastocyanin and cytochrome f interactions in solution.

Most biological functions, including photosynthetic activity, are mediated by protein interactions. The proteins plastocyanin and cytochrome f are reaction partners in a photosynthetic electron transport chain. We designed a 3D computer simulation model of diffusion and interaction of spinach plastocyanin and turnip cytochrome f in solution. It is the first step in simulating the electron transfer from cytochrome f to photosystem 1 in the lumen of thylakoid. The model is multiparticle and it can describe the interaction of several hundreds of proteins. In our model the interacting proteins are represented as rigid bodies with spatial fixed charges. Translational and rotational motion of proteins is the result of the effect of stochastic Brownian force and electrostatic force. The Poisson-Boltzmann formalism is used to determine the electrostatic potential field generated around the proteins. Using this model we studied the kinetic characteristics of plastocyanin-cytochrome f complex formation for plastocyanin mutants at pH 7 and a variety of ionic strength values.

A novel member of the glycosyltransferase family, beta 3 Gn-T2, highly downregulated in invasive human bladder transitional cell carcinomas.

Differential display reverse transcription (DDRT)-polymerase chain reaction (PCR) was used to compare the transcriptomes of invasive and noninvasive fresh human bladder transitional cell carcinomas. A differentially expressed novel gene sharing structural similarity with the human beta 3-galactosyltransferase family, beta-1,3-N-acetylglucosaminyltransferase-T2 (beta 3Gn-T2), was identified. The full-length beta 3Gn-T2 cDNA, containing a complete open reading frame of 1193 bp, was cloned and sequenced. beta 3Gn-T2 exhibited 29-41% homology to the multigene beta 3-galactosyltransferase family. Expression of the full-length beta 3Gn-T2 cDNA in an in vitro coupled transcription/translation assay yielded a primary translation product with an apparent Mr of 46 kDa, which is in agreement with the predicted 397-amino-acid protein encoded by beta 3Gn-T2. Multiple peptide alignment showed several sequence motifs corresponding to putative catalytic domains that are conserved throughout all members of the beta 3-galactosyltransferase family, namely, a type II transmembrane domain, a conserved DxD motif, an N-glycosylation site, and five conserved cysteins. By RT-PCR strong downregulation of beta 3Gn-T2 expression was noted in invasive human bladder transitional cell carcinomas (16 fresh biopsy samples: grade III, T2-T4) compared with their noninvasive counterparts (15 fresh biopsies: grade II, Ta), suggesting that beta 3Gn-T2 may be involved in cancer progression.

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.

High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.

Two-dimensional gel electrophoresis has been the technique of choice for analyzing the protein composition of cell types, tissues and fluids and is a key technology in modern proteomics projects. Here we describe reproducible procedures for running isoelectric focusing and nonequilibrium pH gradient electrophoresis gels that are based on the carrier ampholyte technology originally described by O'Farrell. Moreover, we present a sensitive immunoblotting procedure that has been used routinely in our laboratory to determine the identity of hundreds of human proteins.

Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1.

Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.

Identification of true differentially expressed mRNAs in a pair of human bladder transitional cell carcinomas using an improved differential display procedure.

Differential display in combination with arbitrarily primed polymerase chain reaction (PCR) fingerprinting has become one of the most powerful techniques to identify and isolate mRNAs that are differentially expressed in pairs of biological samples. However, in many cases the cDNA band corresponding to the differentially amplified product contains several cDNA species that comigrate with the cDNA of interest due to the poor resolution of the fingerprinting gels, thus hampering further analysis and identification of the desirable cDNA. To improve the electrophoretic resolution of differentially amplified cDNAs, we have utilized Resolver Gold agarose gel electrophoresis (Ingenius) as an additional step to overcome downstream problems encountered during RNA fingerprinting experiments. To illustrate the power of the modified differential display procedure we present a detailed analysis of the cDNA products differentially displayed in tumor biopsies obtained from a noninvasive (grade II, Ta) and an invasive (grade III, T2-T4) human bladder transitional cell carcinoma (TCC). Several genes that were differentially expressed in this tumor pair were identified. These included: tropomyosin 4, the protein disulfide isomerase precursor (PDI), MRP14, signal transducer CD24, keratins 8 and 13, cytochrome oxidase subunit IV (COXIV), putative transcription factor HOX-1.3, as well as two novel genes of yet unknown function. All of the identified cDNAs were shown to be truly differentially expressed by Northern blotting, reverse transcriptase-PCR (RT-PCR), and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of the corresponding lesions.

A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis.

In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi-bin/celis).

2D protein electrophoresis: can it be perfected?

23 years after O'Farrel developed two-dimensional gel electrophoresis we still debate if the technique can be improved, or if there are other alternative separation technologies that can challenge its central position in proteomic projects. These questions are relevant as the pharmaceutical industry expects proteomic studies to provide novel protein targets for drug discovery and diagnostics. In our opinion, there are various aspects of the technology that can be improved, including resolution, sample preparation and detection, but so far there is no alternative technique(s) available, or any under development, that can replace it.

Rab11a is modified in vivo by isoprenoid geranylgeranyl.

Post-translational adduction of farnesyl or geranylgeranyl moieties to a terminal cysteine residue of proteins is a characteristic feature of ras-related GTP-binding proteins. According to current rules of prenylation, the carboxyl-terminal motif (CXXX or CC/CXC) as well the context of the cysteine residue dictate the extend and specificity of the isoprenoid modification. Rablla, a small GTP-binding protein that is associated with pathways regulating protein traffic, terminates with isoleucine at its C-terminus, suggesting that it may only be geranylgeranylated. Recent finding, however, showed that rab11a can be modified in vitro by different prenyl groups: farnesyl and geranylgeranyl [1]. To determine whether rablla is modified in vivo by both isoprenoids we transiently overexpressed rablla in COS1 cells, and analyzed the translation products by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in combination with metabolic labeling in the presence of either [3H]farnesyl-PP or [3H]geranylgeranyl-PP. Contrary to the in vitro results, our studies showed that rab11a is post-translationally modified in vivo only by geranylgeranyl isoprenoid. The data implied that in vivo there must exist other determinant(s) that are necessary for prenyltransferase recognition.

Human rab11a: transcription, chromosome mapping and effect on the expression levels of host GTP-binding proteins.

Rab11a is a member of the rab-branch of the ras-like small GTP-binding protein superfamily that is associated with both constitutive and regulated secretory pathways. Using a direct procedure for cDNA cloning of small ras-related GTPases, that is based on the screening of eukaryotic cDNA expression libraries using [alpha-32P]GTP as a probe, we have isolated two cDNA clones encoding rab11a. Both clones share identical coding sequences, but differ in the length and sequence of their 3' untranslated regions (3'-UTR). Northern blot hybridisation analysis of various human tissues revealed indeed two mRNA species with lengths of 1.0 and 2.3 kb, respectively. Sequence analysis of the cDNAs identified two different putative polyadenylation signals (AATAAA) at positions 927 and 2302 of the larger transcript. In addition, the 3'-UTR of the larger transcript exhibited several AU-rich elements (ARE) that are believed to control gene expression by regulating the rate of mRNA degradation. Southern blots of human DNA digested with several rare restriction enzymes, and separated by pulse-field gel electrophoresis, yielded the same macro-restriction fragment pattern when hybridised with probes that discriminate between the two transcripts. Taken together, these findings imply that the two mRNA species originate from a single gene, which we have mapped to 15q21.3-q22.31, by the use of different polyadenylation sites. As expected, both rab11a-cDNAs yielded the same protein product when transiently expressed in COS-1 cells, and surprisingly, upregulated the proteome expression profile (de novo synthesis or posttranslational modification of preexisting proteins) of a few other, yet unknown GTP-binding proteins.

Protein abundancy and mRNA levels of the adipocyte-type fatty acid binding protein correlate in non-invasive and invasive bladder transitional cell carcinomas.

The adipocyte type fatty acid-binding protein (A-FABP) is a small molecular weight fatty acid-binding protein whose expression correlates both with the grade of atypia and the stage of bladder transitional cell carcinomas (TCCs). To determine if the protein abundancy correlates with the mRNA levels in non-invasive and invasive lesions, we have analysed fresh TCCs (grade II, Ta; grade III, T2-4) by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and measured the mRNA levels using the reverse transcription linked polymerase chain reaction (RT-PCR). Overall, the results showed a good correlation between protein abundancy and mRNA levels, indicating that the lack of expression of the protein observed in some lesions reflects low levels of transcription of the A-FABP gene rather than translational regulation. In addition, our studies showed that the loss of A-FABP protein observed in some tumors is not compensated by an increase in the skin fatty acid-binding protein PA-FABP, as is the case in the A-FABP knockout mice.

Human and mouse proteomic databases: novel resources in the protein universe.

Proteomics is an emerging area of research of the post-genomic era that deals with the global analysis of gene expression using a plethora of techniques to resolve (high resolution two-dimensional polyacrylamide gel electrophoresis, 2D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting, etc.), quantitate and characterize proteins, as well as to store (comprehensive 2D PAGE databases), communicate and interlink protein and DNA sequence and mapping information from genome projects. Here we review the current status as well as applications of human and mouse proteomic 2D PAGE databases that are being systematically constructed for the global analysis of gene expression in both health and disease (http://biobase.dk/cgi-bin/celis). Furthermore, we discuss the problems one faces when using powerful proteomic technology to study heterogeneous tissue and tumor biopsies, and emphasize the importance of building comprehensive databases that contain a critical mass of information for both known and novel proteins in normal and disease conditions.

Human 2-D PAGE databases for proteome analysis in health and disease: http://biobase.dk/cgi-bin/celis.

Human 2-D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi-bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2-D PAGE reference maps (non-cultured keratinocytes, non-cultured transitional cell carcinomas, MRC-5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2-D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss-Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on-line information is updated regularly.

Identification of isoprenyl modified proteins metabolically labeled with [3H]farnesyl- and [3H]geranylgeranyl-pyrophosphate.

Here we describe a direct approach for two-dimensional (2-D) gel mapping of proteins that are modified by post-translational isoprenylation in mammalian cells. Briefly, transformed human amnion cells (AMA) and transfected COS-1 cells were metabolically labeled with either [3H]farnesyl-pyrophosphate or [3H]geranylgeranyl-pyrophosphate following treatment with lovastatin, which blocks the synthesis of mevalonic acid. The proteins were then separated by 2-D gel electrophoresis and electrotransferred to nitrocellulose filters. The membranes were immersed in dimethyl ether, containing 10% of 2,5-diphenyloxazole prior to fluorography. Over 40 [3H]farnesyl-labeled proteins and over 25 [3H]geranylgeranylated proteins were identified on the 2-D autoradiograms. Several [3H]farnesyl-labeled proteins exhibited the same coordinates (M(r) and pI) as their [3H]geranylgeranylated counterparts, raising the possibility that they may be substrates for both farnesyl and geranylgeranyl transferase(s). The approach offers high resolution of both farnesylated and geranylgeranylated proteins and it may serve as a powerful tool for the identification of hitherto unknown prenylated proteins as well as for the determination of prenylated protein levels, type of isoprenoid modification, and possible changes in protein prenyltransferase activity.

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes.

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.

The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.

The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post-translational modifications. 1082 polypeptides have been identified (protein name, organelle components, etc.) using a procedure or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies, (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry, (v)vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full-length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2-D gel immunoblotting of crude keratinocyte lysates in combination with enhanced chemoluminescence (ECL) detection. Identified proteins are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Ultimately, the aim of the comprehensive database is to gather--through a systematic study of ekeratinocytes--qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Expression of cDNA clones by coupled in vitro transcription/translation and transfection into COS-1 cells: protein mapping in two-dimensional gels.

We present two procedures that can be used to map proteins in two-dimensional gels if the cDNA is at hand. The first procedure, which is illustrated with the expression of cDNAs encoding the fatty acid binding protein 5 (FABP 5), psoriasin and stratifin, makes use of the in vitro transcription/translation assay marketed by Promega. The procedure is simple and allows the mapping of the primary translation product in a very short time. The second method--which faithfully reproduces post-translational modifications--is based on the expression of cDNAs transfected into COS-1 cells using a eukaryotic expression plasmid. This procedure is illustrated with the expression of cDNAs encoding Ha-ras p21 and rab 11, two small GTP-binding proteins known to undergo complex modifications.

A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb.

We report a novel approach for identifying monomeric GTP-binding proteins that is based on probing cDNA expression libraries with [alpha-32P]GTP. In short, a nitrocellulose replica from a plated cDNA expression library is treated with 2% SDS to block the GTP-binding activity of various G proteins expressed by E. coli, thus allowing the direct identification of positive clones. Using this procedure we have cloned several small GTP-binding proteins from human keratinocytes including the human homolog of rheb, a novel member of the ras-related GTP-binding proteins. Human rheb cDNA shares 90% identity with the rat counterpart and it is highly upregulated in transformed human cells of various origin. Northern analysis showed that human rheb is ubiquitously expressed, with the highest levels observed in skeletal and cardiac muscle, and not in brain, as it is the case for rat rheb. The human RHEB gene was mapped to chromosome 10q11.