PROTEOMICS A SEARCH TOOL FOR VACCINES

BY

LAWRENCE OKOROR (Ph.D)DEPARTMENT OF MICROBIOLOGY AND BIOTECHNOLOGY,

WESTERN DELTA UNIVERSITY, OGHARA.DELTA STATE, NIGERIA

INTRODUCTION

*In order to be able to synthesize vaccines successfully a good knowledge of proteomics is necessary. This will help in knowing the proper interaction of the vaccine in the cell.

*The focus of proteomics is a biological group called the proteome. The proteome is dynamic, defined as the set of proteins expressed in a specific cell, given a particular set of conditions. The study of an organism’s complete complement of protein is proteomics.

*The task of studying the proteome has its share of challenges. One involves the sheer number of proteins that need to be identified. The 35,000 genes in the human genome can code for at least ten times as many proteins; in extreme cases a single gene alone can code for over 1,000.

INTRODUCTION CONT’D

*Within a given human proteome, the number of proteins can be as large as 2 million

*Proteins themselves are macromolecules: long chains of amino acids. This amino acid chain is constructed when the cellular machinery of the ribosome translates RNA transcripts from DNA in the cell's nucleus.

*The transfer of information within cells commonly follows this path, from DNA to RNA to protein.

INTRODUCTION CONT’D

INTRO CONT’D

Each level of protein structure is essential to

the finished molecule's function. The primary

sequence of the amino acid chain

determines where secondary structures will

form, as well as the overall shape of the final

3D conformation. The 3D conformation of

each small peptide or subunit determines the

final structure and function of a protein

conglomerate.

CONT’D

There are many different subdivisions of proteomics,

including:

Structural proteomics -- in-depth analysis of protein

structure

Expression proteomics -- analysis of expression and

differential expression of proteins

Interaction proteomics -- analysis of interactions

between proteins to characterize complexes and

determine function.

CONT’D

Proteomics has both a physical laboratory

component and a computational component.

These two parts are often linked together; at

times data derived from laboratory work can

be fed directly into sequence and structure

prediction algorithms. Mass spectrometry of

multiple types is used most frequently for this

purpose.

CONT’D

the study of proteins, however, has been a

scientific focus for a much longer time.

Studying proteins generates insight on how

proteins affect cell processes. Conversely,

this study also investigates how proteins

themselves are affected by cell processes or

the external environment.

Cont’d

Some proteins may not be present in healthy

cells making them useful as target in drug

discovery.

And vaccines are drugs although not

conventional type of drug.

CONT’D

WORKFLOW

LASSA FEVER

Lassa fever is caused by lassa virus which

occurs on epidemic scale all over West

Africa.

It causes close to 350000 deaths annually in

West Africa.

A vaccine was developed against the virus

using the nucleoprotein but it failed to protect

in human but in guinea pigs.

LASSA CONT’D

Lassa virus glycoprotein was

comprehensively studied in order to is

effectiveness for use as a vaccine.

In this regard it comprehensive function was

studied as well as the possible array of

proteins and a possible interaction in human

cell as well as the rat since the rodent is the

reservoir for the virus.

Cont’d

Methods used are

Global sequence alignment

Multiple sequence alignment

Secondary structure prediction which includes hydrophorbicity, transmembrane helices, alpha helices, profile of individual amino acids, which could be done using protscale at expasy, TMpred, PHDtm and prof scale.

Cont’d

A proper set of the windows will definitely

give a good prediction with high score but it

is imperative to start with the default window.

The 3D structure is very important to

determination of the function and its likely

ability to function in the proteome.

Cont’d

Proscan at expasy can be used to determine

the components of the protein

The virulent part of the protein could be

removed by writing a program in python if

after running alignment the removal does not

affect the function, domain repeats could be

carried out to improve the molecular weight

and then antigenicity.

Cont’d

It is also important to see if the molecular

weight of the protein will function properly in

the proteome towards antigenicity and elicit

the production of antibody.

This could be carried out using the multiple

domain repeats which might increase the

molecular weight and thereby antigenicity.

Cont’d

It is also very important run a global

alignment again to see if the domain repeat

has changed the structure of the protein. And

if it will protect against a wide strain of the

virus.

The protein can then be synthesized and

used to vaccinate guinea pigs before human

trials.

Cont’d

Databases like prints, coils and block

databases could also be queried.

Signal peptide is important in determining if

there is an interaction between the protein

and the cell.

A study of the protein-protein interaction is

also important to know how the protein is

interacting with cellular protein.

Cont’d

The type of interaction determines the effect

of the protein in the cell.

Using the scale Hydrophobicity. OMH/Sweet et al., theIndividual values for the 20 amino acids are:

Ala: -0.400 Arg: -0.590 Asn: -0.920 Asp: -1.310 Cys: 0.170

Gln: -0.910 Glu: -1.220 Gly: -0.670 His: -0.640 Ile: 1.250 Leu: 1.220

Lys: -0.670 Met: 1.020 Phe: 1.920 Pro: -0.490 Ser: -0.550 Thr: -0.280

Trp: 0.500 Tyr: 1.670 Val: 0.910 Asx: -1.115 Glx: -1.065 Xaa: 0.000

a. The amino acids with the highest probability of hydrophobicity like phenalanine determine the hydrophobicity of the protein. And the determination of such factor like the hydrophobicity is imperative during vaccine production. It is also important in determining the administration of the eventual vaccine.

Using the scale alpha-helix/Levitt, the individual valuesfor the 20 amino acids are:

Ala: 1.290 Arg: 0.960 Asn: 0.900 Asp: 1.040 Cys: 1.110

Gln: 1.270 Glu: 1.440 Gly: 0.560 His: 1.220 Ile: 0.970 Leu: 1.300 Lys:1.230

Met: 1.470 Phe: 1.070 Pro: 0.520 Ser: 0.820 Thr: 0.820 Trp: 0.990

a. The alpha –helix defines the probability of assigning a helix to the individual

amino acids. And most of the amino acids gave high probabilities. The helical

structure of the protein, and knowing the helical nature of individual amino acids

could help determine the stability of the vaccine to be produced from the protein

especially when domain repeats is to be used to increase the molecular weight

of the of the protein to make it more antigenic.

The Ramanchandran backbone

The hidden markov’s model

Lassa virus glycoprotein 3D structure

Ramancharan backbone

The Ramanchandran backbone of the lassa virus glycoprotein from expasy server. Note the clustering of residues in areas with red colour labeled E, H and B and that most of the exceptions occur in Glycine residues (labeled G). H represents the areas with helix while the L represents the probability of assigning a strand and E is either a strand or a helix. The allowed regions generate standard conformations. A stretch of consecutive residues in the H conformation (typically 6–20 in native states of globular proteins) generates an -helix. Repeating the L conformation generates an extended β -strand. Helices are 'standard' or 'prefabricated' structural pieces that form components of the conformations of most proteins. They are stabilized by relatively weak interactions, hydrogen bonds, between mainchain atoms.

Hidden markov’s model

The hidden markov’s model structure of the protein.

The bars along the diagonal represent the amino

acid type: black bars for non-polar, grey bars for

uncharged polar, no bar for charged side chains,

yellow-green bar for glycine. The upper triangle

shows the contact potential for each pair of

positions, colour red for low energy to blue for high

energy. Hence the lassa virus glycolprotein has high

potential for energy.

3D model

The 3D structure of the lassa virus

glycoprotein using phyre at the expasy

server. The red colour shows area of alpha

helix and are at most 60% conserved. The

blue colour shows area of beta strands which

are least conserved while others are the coil

region. They are also conserved. The coil

regions are also hydrophobic.

Protein interaction

The protein interaction within the cell was

done with genecards and 4 major antigenic

protein was found.

Conclusion

It is imperative to determine the stability of

the vaccine first computationally using the

hidden markov’s model and the

ramanchandran backbone. And later invitro

and invivo.