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.

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.

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.

[Study of protein products of gene expression in cells with altered chromosome sets for genetic mapping]. / Izuchenie belkovykh produktov gennoi ékspressii v kletkakh s izmenennym naborom khromosom dlia geneticheskogo kartirovaniia.

Two-dimensional electrophoresis was used for analyzing proteins in hybrid cells that contained single human chromosomes (chromosome 5, chromosome 21, or chromosomes 5 and 21) against the background of the mouse genome. By comparing the protein patterns of hybrid and parent cells (about 1000 protein fractions for each kind of cell), five fractions among proteins of hybrid cells were supposedly identified as human proteins. The genes of two of them are probably located on chromosome 5, and those of other three, on chromosome 21. Moreover, analysis of proteins in fibroblasts of patients with the cri-du-chat syndrome (5p-) revealed a decrease in the content of two proteins, as compared with those in preparations of diploid fibroblasts. This fact was regarded as evidence that two corresponding genes are located on the short arm of chromosome 5. Methodological problems associated with the use of protein pattern analysis in cells with altered chromosome sets for the purposes of genetic mapping are discussed.

Several small GTP-binding proteins are strongly down-regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype.

High resolution two-dimensional (2-D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP-binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP-binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18,000 to 30,000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP-binding proteins were identified by 2-D gel immunoblotting and these correspond to rap1 and 2 and two forms of rab6. ras-Proteins are most likely present in the [alpha 32P]GTP 2-D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [alpha 32P]GTP-binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.

[Erythrocyte membrane proteins in patients with hereditary spherocytosis]. / Belki membran éritrotsitov u bol'nykh nasledstvennym sferotsitozom.

Red blood cell membrane proteins were studied in a group of patients with hereditary spherocytosis, in comparison with normal donors, to reveal anomalous proteins associated with this disease. For this purpose red blood cells of the patients and normal donors were fractionated, by the age, in Ficoll-400 gradient, as a result red blood cell membranes were obtained with proteins that were investigated by the method of two-dimensional electrophoresis. In comparison of two-dimensional electrophoregrams of red blood cell membrane proteins of normal donors and those of microspherocytosis patients it was found that the latters had additional peptides in the area of glyceraldehyde-3-phosphate hydrogenase and pyruvate kinase. The changes detected in the red blood cell membrane protein composition might be caused by age shifts in the red blood cell population or by the disease type.

Identification of two molecular chaperons (HSX70, HSC70) in mature human erythrocytes.

Two-dimensional gel electrophoresis of cytosolic proteins from mature human erythrocytes combined with immunoblotting revealed the presence of a group of heat shock proteins (HSPs) that included two molecular chaperons of the HSP70 family (HSX70, inducible; HSC70, constitutively expressed) and HSP90. As expected for cells devoid of organelles, erythrocytes do not contain stress proteins that are localized either in the mitochondria (HSP60, glucose-regulated protein (GRP 75) or in the endoplasmic reticulum (GRP78 or Ig heavy chain-binding protein, endoplasmin). Since red cells are unable to replace proteins whose structure has been damaged by environmental changes the results are taken to imply a role for chaperons in monitoring, protecting, and maintaining the structure and stability of erythrocyte proteins.

[Comparative study of human erythrocyte membranes in normal people and in Huntington's chorea patients]. / Sravnitel'noe izuchenie membran éritrotsitov krovi chekoveka v norme ibol'nykh khoreei gentingtona.

Erythrocytes of healthy volunteers and of patients with hereditary chorea were studied. Evaluation of the state of cellular membrane was carried out by measuring osmotic resistance, activity of Na+, K(+)-ATPase and protein composition. In the patients osmotic resistance of erythrocytes was distinctly decreased down to 66.3 +/- 3.3, while the Na+, K(+)-ATPase activity was increased 4-fold as compared with controls. Protein composition of erythrocyte membranes, studied by means of two-dimensional electrophoresis, was similar both in healthy persons and in patients with hereditary chorea when the electrophoretograms were analyzed visually. An additional protein with Mr = 30,000 and r-1-0.25 was detected in one of the patients. Slowly sedimenting fraction of erythrocytes was found in almost all the patients with hereditary chorea when erythrocytes aging was studied by means of fractionation in Ficoll density gradient. The fraction was not observed in healthy persons. These data suggest that the cell membranes in Huntington's chorea are altered as compared with normal state.

Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance.

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis ("self-digestion") involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.

[Characteristics of endogenous proteolysis in preparations of human erythrocyte membranes]. / Kharakteristika éndogennogo proteoliza v preparatakh membran éritrotsitov cheloveka.

Endoproteolytic activity in human erythrocyte membrane preparations has been examined at 37 degrees C by one- and two-dimensional electrophoresis. Two-dimensional mapping has shown that the presence of leukocyte enzymes in erythrocytes prepared in a regular manner (centrifugation) cannot be excluded. Sedimentation in the 1.5% dextran 500,000 with the following erythrocyte purification on HBS-cellulose has made it possible to prepare erythrocyte membranes characterized by low level endoproteolytic activity without leukocyte enzymes. The marker peptide has been found. It is likely to be a specific product of the enzyme activity of membrane localization.

[Two-dimensional map of membrane proteins from human erythrocytes]. / Dvumernaia karta membrannykh belkov éritrotsitov cheloveka.

Human erythrocyte membrane proteins were analyzed by a modified two-dimensional electrophoresis performed according to O'Farrell. This method was used to construct a two-dimensional map of human erythrocyte membrane proteins. The map plotted in the coordinates "relative molecular mass versus relative electrophoretic mobility during IEF" was used for the characterization of 189 proteins. The position of major membrane proteins in the map was determined on the basis of their Mr, pI as well as literature data. Carboanhydrase was positioned by coelectrophoresis. A comparative analysis of erythrocyte membrane and cytosol preparations by two-dimensional protein mapping revealed that some of erythrocyte proteins have dual localization.

[Two-dimensional electrophoresis of human erythrocyte membrane proteins in the presence of triton X-305]. / Dvumernyi élektroforez éritrotsitarnykh membrannykh belkov cheloveka v prisutstvii tritona X-305.

Highly effective method of proteins fractionation--two-dimensional electrophoresis by O'Farell is now widely used in biochemical and genetic studies. Membranes of erythrocytes are used for detection of firmly bound markers among their protein components for development of suitable diagnostic tests.

[Analysis of the results of two-dimensional electrophoretic fractionation of proteins using a system of digital processing of video information]. / Analiz rezul'tatov dvumernogo élektroforeticheskogo fraktsionirovaniia belkov na komplekse tsifrovoi obrabotki videoinformatsii (SVIT).

Possible solution of four main problem in analysis of two-dimensional electrophoregrams using the complex set for processing of video-information SVIT is considered: I. a. potentiality for evaluation of correlation between integral optic density of spots on gel plates and amount of protein fractionated, 2. qualitative comparison of similar spots from various electrophoregrams, 3. the problem of overlapping spots, where the plane pictures of spots were transformed into three-dimensional system of peaks, 4. use of bench-marks for geometric transformations and production of unified pictures. The complex set or its modifications were able to settle these problems.

[Study of human erythrocyte membrane proteins by 2-dimensional electrophoresis]. / Izuchenie belkov membran éritrotsitov cheloveka metodom dvukhmernogo élektroforeza.

Fractionation of human erythrocyte membrane proteins was performed using a modification of two-dimensional gel electrophoresis described by P. O'Farrel with isoelectric point plotted against molecular mass. All major erythrocyte proteins, including high molecular weight proteins, such as spectrin and band 3 protein, identified by one-dimensional sodium dodecyl sulfate gel electrophoresis, were visualized by silver staining of two-dimensional gels. All in all about 50 polypeptides were distinguished on two-dimensional electrophoretic patterns. Preliminary protein map was developed.

[Detection of the interstrain polymorphism of heart muscle proteins in mice using two-dimensional electrophoresis]. / Vyiavlenie mezhlineinogo polimorfizma belkov serdechnoi myshtsy myshei metodom dvumernogo élektroforeza.

Cardiac muscle proteins of four inbred murine strains were analyzed by one- and two-dimensional electrophoresis. Of those, 27 and 161 protein fractions were characterized in terms of molecular mass and relative electrophoretic mobility. The protein fractions were identified as corresponding to creatine phosphokinase, myoglobin and an albumin-like protein. Six polypeptides characterized by interlinear polymorphism were identified.

[Active form of dihydropteridine reductase in human chorion cells. Possibility of prenatal diagnosis]. / Aktivnaia forma digidropteridinreduktazy v kletkakh khoriona cheloveka. Vozmozhnost' prenatal'noi diagnostiki.

Activity of dihydropteridine reductase was studied in human chorion cells for development of accurate methods for prenatal diagnosis of phenylketonuria (especially its most severe form known as lethal hyperphenylalaninemia).

[Effect of the supranucleosomal chromatin organization on histone-DNA interrelations]. / Vliianie nadnukleosomnoi organizatsii khromatina na vzaimootnosheniia gistonov i DNK.

It has been demonstrated by the method of competitive displacement of own chromatin histone by excess total histone that chromatin dispersity influence the strength of histone-DNA interactions in a medium of physiological ionic strength. Histone NI was removed from chromatin after the quantity of total histone added to chromatin was equivalent to that existing in chromatin. The proportion of histones H2A and H2B removed from chromatin was increased after mechanical of ultrasonic degradation of chromatin at 5-20-fold excess of total extra-histone. In some histone preparations, the removal of histones H2A and H2B was not detectable at even 200-fold excess of total histone. This may be explained by strengthening histone-DNA interactions in superhelical loops of chromatin.

[Electrophoretic analysis of the characteristics of the protein composition of leukocytes from Down's syndrome patients]. / Elektroforeticheskii analiz osobennostei belkovogo sostava leikotsitov bol'nykh s sindromom Dauna.

Protein with a molecular mass of 53000 daltons undetectable in healthy persons was identified by electrophoresis in peripheral blood leukocytes of patients with Down's syndrome. The protein was completely extracted with 0.4 N HCl from leukocyte homogenates and was found to be identical, as regards electrophoretic mobility, to protein detected in two patients with chronic myeloblastic leukemia. The causes of discrepancy between theoretically expected and electrophoresis-revealed differences in protein composition of normal and trisomal cells.