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Proteomics & Genomics fight H5N1, bird flu virus - Amit Kulshresth, Dhanya Nair (Feb 18,2006)

Synopsis : This article and illustrations seek to explain the application of Proteomics and Genomics to combating the H5N1, avian influenza virus. A brief description of both fields precedes an explanation of gene expression. H5N1 protein structure clarifies which protein causes infection and then the applications of latest technologies combating H5N1 are detailed.

Genomics : Genomics studies the entire DNA sequence of an organism's genome and the use of the genes. It refers to the study of all of the nucleotide sequences, including structural genes, regulatory sequences, and non-coding DNA segments, in the chromosomes of an organism. The goal is to find all the genes within each genome, find what genes do, how they are controlled, and how they work together.
It looks at all the genes as a dynamic system, over time, to determine how they interact and their physiology, in global manner.

It uses this information to understand molecular mechanisms of disease, answer scientific questions and to develop improved medicines.

Proteomics :
Proteomics is the study of structures and functions of proteins. It is different from and more complex than genomics. It can be understood as proteins expressed by a genome.

Proteomics includes the identification, abundance, spatial distribution and quantification of proteins, but also determines their localization, modifications, interactions, activities, and their function. With the sequencing of genomes of many biological organisms, the scope of Proteomics has broadened from protein identification and characterization to analyzing the protein structure, function and protein-protein interactions.

Identifying proteins: To identify proteins, x-ray crystallography; nuclear magnetic resonance (NMR) spectroscopy are being used. Protein arrays are also being used; they consist of a library of hundreds or even thousands of different proteins spotted individually in a known location on a chip. It uses the proteins themselves for studying the protein/ protein interactions or of probes (often antibodies) for capturing proteins.
Protein arrays are more difficult to work with than DNA arrays as proteins vary hugely in their chemical nature. They also bind to each other in several ways. In contrast, DNA fragments, only the nucleotide sequence varies and they pair by simple Watson-Crick base method.

Problem in Proteomics and Genomics: Proteomics can overlook proteins that are present in minute quantities, and genomics does not indicate the extent to which transcripts are translated into proteins. Companies are now producing plasma Immunodepletion Kit, that removes about 99% of twenty high abundant plasma proteins, thereby making it easier to visualize low-abundance proteins in samples.

Comparison between Proteomics and Genomics :

Refers to all the proteins produced by an organism Refers to the entire set of genes in an organism (haploid set)

Proteins are responsible for both the structure and the functions of all living things, life is due to proteins. Usage:

  1. Catalyze enzymes
  2. As messengers- neurotransmitters
  3. Regulate cell reproduction
  4. Defensive action (antibodies)
  5. Tissue growth
  6. Oxygens transport
Genes are the instructions for making proteins (Genes-mRNA-Proteins).
Proteome varies from one cell type to another in an organism although all have the same genome. It varies in RBC to WBC to neuron, fibroblast, It also varies with health or disease and also with time for a cell during its lifecycle as it responds to environment i.e. new nutrients, hormones, foreign bodies etc. Genome remains unchanged from one cell to another (except for mutations or un-repaired damage to its DNA)
Complex: A single gene can give rise to a number of different proteins due to formation of glycoproteins, addition of phosphates to amino acids, RNA editing Relatively simple and constant
Humans : 400,000 proteins (each having different functions) made by genes Humans : 22-23000 genes Virus : 3- 200 genes

Gene to proteins:

A gene is a section of genome that carries information for the construction of a protein.

A protein is constructed by joining a sequence of amino acids. The precise amino acid content, and the sequence of those amino acids, of a specific protein, is determined by the sequence of the bases in the gene that encodes that protein. Thus in short the sequence of amino acids present in a protein is compiled corresponding to the sequence of bases of a gene.

The path leading from genes to proteins involves two steps called transcription and translation.

Transcription takes place in the nucleus of the cell. In this step, the gene of the genome molecule is copied to a strand of RNA molecule called Messenger RNA (mRNA).Thus translation is the process of converting the mRNA codon sequences into an amino acid sequence.

Translation is the step that finally converts the copied code of mRNA to proteins. The process of protein synthesis takes place at the site of ribosomes that are present in the cytoplasm of the cell.

gene expression to create protein

In this process mRNA leaves the nucleus, travels to the site of ribosomes. Here at this site as per the code, amino acids are collected and linked together to form the appropriate protein molecule.

Ribosomal RNA and Transfer RNA assist the process of translation.
Messenger RNA (mRNA) is used for construction of a protein.
Ribosomal RNA (rRNA) is the construction site where the protein is made. Transfer RNA (tRNA) is the truck delivering the proper amino acid to the site at the right time.

Amino acids link up in a chain format to form large molecules called proteins. There are 20 different amino acids. They combine in different combinations to form thousands of proteins. The order of amino acids in the protein molecule determines its structure and function. The final protein molecule may consist of several hundred amino acids linked together according to the instructions encoded in the mRNA. In addition, each protein can undergo a variety of post-translational modifications that further influence its shape and functions. Proteins may serve e.g., as enzymes, hormones or structural components of a cell.

H5N1 (bird flu virus) & its proteins:

The H5N1 infects a human or bird using its proteins on its envelope, inhibiting its proteins could curtail the infection from occurring or spreading.

However in the replication process mutations or recombination occurs, altering its genome sequence sufficiently enough to create a new set of proteins, for example the H5N1 virus can now infected the ciliated cells of bird, humans and pigs though structurally they are different.

Lets look at its major proteins, some of them have been described below, and refer the animation for more details.

H5N1 virus and its protein structure

Haemagglutinin (H or HA): These spike like projections present on the envelope, help the virus recognize and attach itself to the cells of the host. .They are made up of carbohydrate & protein complexes. Haemagglutinin is a 135Å trimer, that binds to mucoproteins (found on the surface of epithelial cells) containing terminal N-acetyl neuraminic acid. This protein is also responsible for membrane fusion in virus entry.

N : Neuraminidase (N or NA ): These spike like projections, made up of carbohydrate & protein complexes, present on the envelope allows newly formed virus particles to break free from their host cells. It is crucial to this budding and release process. It is a 60Å tetramer. The nucleic acid in A and B types are arranged in 8 segments (7 for influenza type C). Each segment comprises of single strand, negative sense RNA2 associated with three polymerase polypeptides giving RNP.

Ion channel M2 : pH activated ion channels (M2) are involved in the uncoating process of the virus.

M1 ( Matrix protein) : In the virus particle, the vRNPs are connected to each other and with the viral envelope by the matrix protein,

NS1: Anti-interferon protein. Effects on cellular RNA transport

H5N1 virus protein structure and description

Using Proteomics/Genomics to fight H5N1

The problem is that every time an animal is infected, H5N1 virus gets an opportunity to mutate its genetic code. It is in a process of rapid evolution, at present four variants of it are in circulation.

Drug development (Oseltamivir or Tamiflu): Identification of the structure of viral protein helps in creating the drugs. The drug Oseltamivir works by decreasing the ability of influenza virus to spread from infected cells to uninfected cells by inhibiting neuraminidase, the protein required for the virus to exit infected cells.

However a recent study has shown that Oseltamivir has to be used for eight days in mice to suppress the virus, the virus continues to grow if the drug is stopped after five days.

By identification of NS1 Protein: In Jan 26, 2006, scientists were able to identify through prototyping, a wide analysis of the genome sequences of different virus strains (avian, swine, and human influenza sequencing data), a unique protein responsible for the high mortality of H5N1. The protein NS1 (having a bird origin) works in combination with other avian flu proteins, within the infected cells and disrupts key cellular processes in humans.

Viruses in which the entire complements of influenza genes, including NS, were derived from an avian source caused the recent H5, H7 and H9 outbreaks in Asia. Thus the introduction of avian NS1 into human cells can potentially disrupt many cell pathways...while the human NS1 does not.

The protein NS1 might become a key target for future anti-flu drugs.

By CombiMatrix DNA arrays (using genomics): CombiMatrix group has made commercially available the first microarray designed for the H5N1 "Bird Flu" influenza A virus. The conventional approach to materials discovery involved successive cycles of synthesis and testing, making the process costly, labor and time intensive. The new approach is speedier and more economical as it enables the synthesis of many thousands of materials in parallel followed by testing of all of those materials at one go.

CombiMatrix's DNA microarray employs the parallel synthesis of large numbers of nano-structured materials. These materials are tested using the chip-based technology. It not only allows for the identification of the H5N1 virus but also the tracking of mutations as the virus moves from host to host. The chip measures about 22 mm by 5 mm. Below it is one of the thousands of microelectrodes per chip where DNA is synthesized and controlled by the digital circuitry. The arrays of oligonucleotides capture molecules synthesized on the chip for use in Genomic and Proteomic applications.

By PCR based test (using genomics) : Roche Diagnostics and Tib Molbiol, Berlin, Germany, announced the worldwide availability of a new PCR-based test designed to allow researchers detect the influenza A virus H5N1 in birds.

It uses a technology platform that is commonly available in research laboratories. It allows for identification of the Influenza A H5N1 subtype within hours compared to days with many other methods. The test also detects genetic material of the virus instead of proteins (antibodies) that are identified by other immunological tests currently used.

By Direct RNAi™ (using both) : Alnylam Pharmaceuticals and University of Georgia jointly will work to discover short interfering RNAs (siRNAs) to target key flu genes required for virus replication.

The process of DNA -- RNA - protein at times produces abnormal proteins causing human disease. RNAi inhibits the production of abnormal proteins by gene silencing (through silencing mRNAs).

Many of the best-selling drugs today either act by targeting proteins or are proteins themselves. Advances in proteomics are helping scientists create medications that are "customized", more effective and have fewer side effects.



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