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The research program of the center organized on the basis of the
V.N.Orekhovich
Institute of Biomedical Chemistry, RAMS
Owing to concerted effort by numerous state and commercial structures, the
human genome has completely been sequenced in 2000. All 24 human
chromosomes are mapped, the defects of hundreds of genes responsible for
the development of hereditary diseases have been revealed. A new medical
concept has been advanced whereby all diseases can be divided into two
major groups: (a) hereditary diseases - a consequence of transmission of
defective genes from parents to their children and (b) nonheritable or so
called socially-significant diseases, which make up more than 95% of all
human diseases and result from disturbances in normal genes' expression
regulation. Thus, all human diseases, this way or another, are associated
with the genome; the only difference is that the diseases of the former
group are due to a defect(s) in gene's structure while those of the latter
group are caused by disturbances in gene expression's regulation. The
cataloguing of human genes is an achievement which can hardly be
overestimated: for many years to come this catalogization will serve as a
basis for the development of basic biochemistry, molecular biology and
genome-associated applied sciences. The future of this area of research
called "genomics" is even more brilliant. Currently the emphasis
has swung from sequencing the human genome to sequencing the genomes of
animals and microorganisms, particularly pathogenic, and plant genomes.
These researches promise enormous achievements in medicine, especially in
the struggle against infectious diseases and in agriculture.
While discussing the subject of genomics, we are approaching another
topic, namely, what is the postgenomic era?
How the informational structure (gene) is connected with the actually
working molecular machine (protein) ? To answer this question, it has to
be taken into account that in those few works, where the map of mRNA was
compared with the proteinous map within the same cellular system, no
strict correlation between the two maps was revealed.
Thus, the informational knowledge cannot be directly converted into the
knowledge of actually operating protein molecules. As a result, a new area
of research has appeared which is called "proteomics" and which
deals with inventory of proteins. At first glance this is an utterly
impossible task. While the human genome map is the same for all human
cells (24 chromosomes), in the proteomic map each cell is individual.
Although the cell may have only 30 - 70 thousands of functional genes, the
numerous modification reactions may increase the number of proteins in it
up to several millions. There are two definitions for proteomics: a narrow
one (the so-called structural proteomics) and a broader one, encompassing
both the structural and functional proteomics.
In a narrow sense of the word, "proteomics" is a science dealing
with cataloguing of proteins based on a combination of several methods:
2D-electrophoresis, mass-spectrometric analysis of molecular mass,
sequencing of electrophoretically-divided proteinous bilogical material
with subsequent analysis of the results obtained by use of bioinformatical
methods.
In a broader sense, the terms "proteome analysis" or
""proteomics" can be used not only for cataloguing proteins
of a biological subject but also for the monitoring of reversible post-translational
modification of proteins by specific enzymes, i.e. phosphorylation,
glycosylation, acylation, phrenylation, sulfurization, etc.
To date, more than 300 different types of post-translational modification
have been characterized with the aid of proteomics. Another aspect of
functional proteomics is the clarification of a composition of
functionally active complexes forming different metabolic chains and,
also, determination of interactions between various proteins or subunits
of oligomeric complexes by a combination of methods for isolation of these
complexes with subsequent mass-spectrometric analysis. Lately, structural
proteomics is often called expressional proteomics while functional
proteomics is also designated as cell-mapping proteomics, since it
elucidates the interactions of proteins within metabolic pathways.
In our country on the basis of the V.N.Orekhovich Research Institute of
Biomedical Chemistry (IBMCh) of RAMS a center for proteomic researches,
the first in Russia, is being organized. The scientific program of the
center comprises two major directions which will be developed in the
proteomic department of IBMC in cooperation with other institutes.
The first direction involves basic researches in the field of proteomics;
they will be aimed at creation of proteomic maps of proteins for various
biological membranes. Up to now, the problem of effective analysis of
membrane proteins by use of 2D-electrophoresis and mass-spectrometry
remains unsolved. All major achievements in proteomics are related to the
analysis of water-soluble proteins. Separation of membrane proteins and
their analysis is a much more difficult task. Given that the membrane
proteins constitute up to 30% of all proteins in the genome and that they
are highly significant for signal systems, transport processes and
oxidative reactions, the necessity of solution of this problem is beyond
doubt. As membrane proteins, will be studied the proteins of erythrocyte
membranes, of microsomal membranes, of mycoplasm membrane, and those of
microbial and plant cells. Until now, there were serious methodological
limitations for fractionation by 2D electrophoresis of hydrophobic
proteins, which are known to require detergents for their solubilization.
At the same time, the first reports have appeared in the literature about
successful application of (alternative to 2D) multi-dimensional
chromatographic techniques, enabling successfull separation of membrane
proteins.
The research program of the proteomic center presupposes the application
of various technologies for membrane proteins' fractionation to construct
proteomic membrane maps. The first step in the implementation of this
program will comprise (a) fractionation of membrane proteins A.Laidlawii
by use of the non-ionic detergent, triton X-114, with subsequent
two-dimensional separation of fractions by 2D electrophoresis and (b)
mass-spectrometric analysis of erythrocyte membrane proteins (the problem
of 2D electrophoresis of these proteins has so far been successfully
resolved). Simultaneously, the studies will be carried out on construction
of proteomic maps of lymphocytes and of the liver cell microsomal
fraction. The main object of study upon construction of their proteomes
will be the cytochromes P-450 isoforms. Study of the regularities of
expression of these monooxygenases under the influence on laboratory
animals (mico) of pharmacologically active substances and toxic compounds
will provide a better understanding of the mechanism of complex response
of cytochrome genes to xenobiotics application. The analysis of the
proteomic maps of human lymphocytes will enable to use in
pharmacoproteomic studies the data on cytochrome P450 isoforms' content in
these latter cells.
The combination of up-to-date biosensoric and mass-spectrometric
technologies by the "fishing" approach will make it possible to
elucidate the contact sites of interacting molecules in the process of
their functioning. It will yield new data on the mechanism of protein
molecules' interaction, and thus will make a valuable contribution to the
solution of the protein-protein recognition problem - one of the basic
problems of modern biochemistry and molecular biology.
Based on the analysis of proteomes of normal and pathologically altered
cells, as well as on the studies of drug-sensitive and drug-resistant
microbial cells, the distinctions in their composition will be revealed.
On the basis of these distinctions, new protein targets will be proposed
for which, with the aid of computer modelling, appropriate ligands with
high affinity to these targets will be found, which could later be used
for subsequent synthesis of new pharmaceuticals.
One essential requirement for the rational engineering of pharmaceuticals
and diagnosticums, for the elaboration of biotechnological processes, i.e.
for addressing the problems outlined in points 1 - 4 of the present
project, is the knowledge of spatial structure of proteins playing a
crucial role in the given disease state or the given biotechnological
process. The most effective method (now in use) for determination of
spatial structure of proteins in near-normal physiological conditions is
the nuclear magnetic resonance spectroscopy (NMR).
The interactions of target proteins with ligands as well as
protein-protein interactions will also be investigated - by use of high
resolution NMR.
Modern NMR methods in powerful magnetic fields open up unique
opportunities for determination of spatial protein structures in
solutions, for establishing structures of enzymes' active centers,
elucidating the mechanisms of their binding to substrates and inhibitors,
calculating the stability constants for complexes formed therewith and
investigating the dynamics of both the individual proteins and the
supermolecular assemblies emerging upon protein-protein interactions in
solutions. In the studies to be conducted, the methods of two- and three-
dimensional NMR spectroscopy will be employed in combination with
relaxation measurements and dynamic NMR methods in a wide range of
temperatures and concentrations and at various pH values. The quantitative
information about the thermodynamics of protein-ligand and protein-protein
interactions, incuding the competitive binding data, will be compared with
the results of computer modelling and thus form the basis for selection
and modification of potential pharmaceuticals. In the course of this work,
new computer programs will be created for treatment of dynamic NMR spectra
and for investigation of the kinetics of complex chemical equilibriums,
including several successively-parallel routes. In carrying out NMR
experiments, a wide application of recombinant labeled ligands is planned.
A characteristic feature of researches to be carried out in the Center
will be, from the very outset, their commercialization. Proteomics will be
used for creation of new diagnostic methods. Now, this trend has got top
priority the world over.
1. Medical
proteomics-based diagnostics.
Applied researches in
medical diagnostics will be developed in several directions: (a)
Construction of proteomic maps of serum and amniotic fluid for the purpose
of complex determination of foetus pathology markers. Special attention
will be given to elucidation of etiological mechanisms of fertility, to
pathogenesis of chronic miscarriage and elaboration of algorithms for the
complex therapy of these states based on dynamics of specific markers'
alterations. The first step presupposes elaboration of assays for
determination of AMGF (glycodelin), alpha-fetoprotein, g-XGH, lipokalins,
SA-125, the (insulin-like growth factor-1)-binding protein (IGF BP1),
trophoblastic g-globulin, placental lactogen, pregnancy-associated
alpha-2-microglobulin, relaxin. The second step involves elaboration of
methods for identification of new miscarriage markers, such as HLA-G
antigen, tissue- and organ-specific autoantibodies and so forth. Special
studies presuppose elucidation of specific features of posttranslational
modification of basic markers for pathological processes, leading to
hormonal background alterations, as well as determination of
pharmacogenetic approaches to correction of these indices;
(b) Diagnostics of tumors. Special attention will be given to detection of
markers for the so-called "silent tumors" of bone tissue,
ovaries and others. In particular, the determination of protooncogen-encoded
proteins, such as myc, scr, fos, jun, myb, fmc, raf-1, will be carried
out. Also to be studied are the possibilities of complex determination of
integrins, endothelial kadkherin, vinkulin, E-selectin and some other
protooncogens. The proteomic maps of tumor tissues will be constructed for
diagnostic purposes, and the results obtained are supposed to be used for
early diagnosis and for treatment optimization;
(c) Diagnostics of hepatitis. Particular attention will be given to the
elaboration of methods for the diagnostics of hepatitis, in whose
pathogenesis the autoimmune reactions play the leading role. Lack of
domestic sets of reagents for definition of autoimmune hepatitis markers
makes this part of the research very timely for application of proteomic
technologies to practical health care. Also, the attempts will be made to
reveal the proteins of B and C hepatitis viruses in the serum of patients
with hepatites - both for the diagnosis of these diseases and for the
control of treatment efficacy.
2. Proteomics of
industrial microorganisms
Bacteria are living
organisms of the most elementary structure, capable of independent
existence in the environment. It may be stated with certainty that
bacteria provide a perfect model for basic researches in the post-genomic
era because (a) their structure is very simple; (b) their genome has been
fully decoded (more than 40 bacterial species by the end of 2000); (c)
there is a wealth of data on their genetics, biochemistry, molecular
biology, and structural organization; and, lastly (d) these organisms
offer an ability to monitor their growth under strictly controlled
conditions.
1. With regard to the practically oriented basic researches, it would be
worth-while to dwell, within the framework of this program, on the two
most studied bacterial species - E.coli and B.subtilis. These species of
bacteria are widely used in industrial production: B.subtilis is used for
obtaining of enzymes, nucleotides, vitamins; E.coli, for production of
human proteins (insulin, interferon and many others) and for production of
amino acids, carotinoids and others.
From a practical viewpoint, microbiological industry requires the
optimization of three parameters: 1) high speed of growth; 2) high yield
of crude target product; 3) safety of strain for the man and the
environment.
Based on genomics and bioinformatics, the proteomics, especially in
combination with transcriptomics and isotope methods of measurement of
metabolic flows, may form a scientific basis for solution of above
mentioned practical problems, thereby bringing us nearer to the main aim
of biology - quantitative and adequate description of living organism
behavior at the molecular level.
For the program performance it is planned to resolve the following
problems:
1. To determine a set of E.coli genes, which are never expressed in
laboratory conditions. To delete several genes of these species and to
show that such deletions have no effect on technological characteristics
of strains. Towards the end of this term or in subsequent years to delete
10 to 15% of the E.coli genome. These mini-bacteria must grow better,
spend lesser amounts of substrate for biomass synthesis but not survive in
the environment.
2. To study the E.coli proteome in the phase of culture transition from
the logarithmic phase of growth to the stationary phase. To characterize
the expression of gene groups involved in central metabolism. To detect
the groups of genes regulated by common regulation systems. Using the data
base, to obtain indications to target genes, whose changes may prolong the
productive stage of biosynthesis (without cell division).
3.Construction of proteomes of tubercular pathogens and creation on
their basis of new preparations to overcome multiple resistance of
mycobacteria to medicinal preparations
At present, the spreading of tuberculosis in Russia has acquired menacing
proportions. High incidence rate of tuberculosis is connected with the
lowered efficiency of traditional anti-tuberculous medicines and spreading
of multi-resistant pathogenic strains of M.tuberculosis. Present-day
tuberculosis is characterized by a severe disease course with quick
formation of cavities and caseous decay. According to the data of the
Research Institute of Phythisiopulmonology of the I.M. Sechenov Moscow
Medical Academy, primary drug resistance is observed, on average, in 35%
patients.
One of the cardinal ways to overcome drug resistance of mycobacteria
consists in application of intensively developing proteomic analysis.
This approach is optimal with respect to: identification of genes,
determination of appropriate macromolecular components of bacterial cells
responsible for resistance, revelation of new genes and virulency factors,
performance of exhaustive comparative analysis of resistant strains and
creation, on their basis, of data bases providing the estimation and
prediction of resistance to antituberculous medicines.
At present, the DNA sequence (consisting of 3924 genes) of the M.
tuberculosis H37Rv genome is completely decoded. Fractionation and partial
functional identification of 1800 proteins of virulent strains have been
performed. At the same time, it is necessary to admit that the currently
available data on genes and mycobacteria's virulency factors remain scarce
and fragmentary. One very important goal of proteomic analysis is the
study of those genes, encoding the virulency factors; the aim is to allow
the mycobacteria to neutralize, parially or fully, the influence of
mechanisms underlying the non-specific resistance and/or immunity.
4.Proteomic analysis of
trangenic plants expressing epitopes of antigenes to viruses of human
hepatitis B, C and E
Of great importance for the
genetics, molecular biology and biotechnology of plants are the researches
on genomics of a model flowering plant, arabidopsis. Knowledge of
arabidopsis' genes will provide a better insight into the basic processes
of reproduction and realization of genetic information in high plants and,
in particular, will lead to creation of a new generation of agricultural
products and plants for medical use. At present, the development of these
researches is impossible without detailed investigation of plant cell
proteins constituting its proteome. Modern high-precision methods of
mapping, visualization of proteins and study of protein-protein
interactions allow the identification of minor qualitative and
quantitative changes in cell proteomes, arising under various outer
influences and in physiological processes. Special importance these
researches have acquired after the creation of transgenic agricultural
products - for which they serve as an important certification criterion.
Among the most topical researches are those employing plants for creation
of new vaccines against viruses of hepatites B, C and E. The preparations
obtained may be used as "eatable" vaccines and this will make
their production and application significantly cheaper. Such researches
are currently being conducted on tobacco and arabidopsis, but in future
the elaborated techniques will be extended to such cultures as cabbage,
rape, tomatoes. Since these plants are closely related to arabidopsis, one
can assume that the proteomic analysis of the model plant systems under
study will enable to effectively use the obtained results for closely
related technical cultures as well. |