In-Silico Analysis to identify Non- synonymus SNPs in SLE (Systemic Lupus Erythematosus) gene , IL2

(Interleukin- 2 )MEMBERS: RUCHIKA KUMAR(15MSB0001)

SOUMILI DAS GUPTA(15MSB0059)ANJU.G(15MSB0062)

SNEHA DUTTA(15MSB0023)

SYSTEMIC LUPUS ERYTHEMATOSUSSystemic lupus erythematosus (SLE), also

known as lupus, is an autoimmune disease The immune system normally fights off

dangerous infections and bacteria to keep the body healthy

An autoimmune disease occurs when the immune system attacks the body due to it confuses for something as foreign

There are many autoimmune diseases, including systemic lupus erythematosus (SLE)

SLE is a chronic disease that can have phases of worsening symptoms that alternate with periods of mild symptoms

According to the Lupus Foundation of America, at least 1.5 million Americans are living with diagnosed lupus

The foundation believes that the number of people who actually have the condition is much higher and that many cases go undiagnosed.

SYMPTOMSSymptoms can vary and can change over time. Common symptoms include: Severe fatigue Joint pain Joint swelling Headaches Rash on the cheeks and nose, which is called “butterfly

rash” Hair loss Anemia Blood-clotting problems Fingers turning white or blue and tingling when cold,

which is known as Raynaud’s phenomenon.

CAUSES OF SLE The exact cause of SLE isn’t known, but several factors have been

associated with the disease. The disease isn’t linked to a certain gene, but people with lupus often

have family members with other autoimmune condition. Environmental triggers can include:• Ultraviolet rays• Certain medications• Viruses• Physical or emotional stress• Trauma• Certain hormones SLE affects women more than men. Women also may experience more

severe symptoms during pregnancy and with their menstruation. Both of these observations have led some medical professionals to

believe that the female hormone estrogen may play a role in causing SLE. However, more research is still needed to prove this theory.

DIAGNOSIS AND TREATMENT Signs and symptoms of lupus o Sun sensitivity rashes, such as a malar or butterfly rash mucous

membrane ulcers, which may occur in the mouth or nose,o Arthritis, which is swelling or tenderness of the small joints of the

hands, feet, knees, and wrists o Hair loss hair thinning signs of cardiac or lung involvement, such

as murmurs, rubs, or irregular heartbeats No single test is diagnostic for SLE. Treatment for SLE No cure for SLE exists. The goal of treatment is to ease symptoms.

Treatment can vary depending on how severe your symptoms are and which parts of your body SLE affects. It include:

o anti-inflammatory medications for joint pain and stiffnesso steroid creams for rasheso corticosteroids to minimize the immune responseo antimalarial drugs for skin and joint problems

EFFECT OF SLE SLE can damage or cause complications in

systems throughout your body. Possible complications may include:

o blood clots and inflammation of blood vessels or vasculitis inflammation of the heart, or pericarditis, heart attack, stroke , memory changes, behavioral changes seizures

o inflammation of lung tissue and the lining of the lung, or pleuritis , kidney inflammation, decreased kidney function, kidney failure

SLE can have serious negative effects on your body during pregnancy. It can lead to pregnancy complications and even miscarriage.

The IL-2 Defect in Systemic Lupus Erythematosus Disease has an Expansive Effect on Host Immunity

IL-2 production is decreased in systemic lupus erythematosus (SLE) patients and affects T cell function and other aspects of host immunity

Transcription factors regulating IL-2 that causes dysregulation (IL-15 and IL-21) which are abnormally expressed in SLE

Decreased IL-2 production in SLE patients leads to many immune defects such as decreased IL2 production, decreased activation-induced cell death (AICD), and decreased cytotoxicity

IL-2 deficiency results in systemic dysregulation of host immune responses in patients suffering from SLE disease

Defective IL-2 Production in SLE

IL-2 production is upregulated through an autocrine (or paracrine) loop due to this defect in the T cell receptor (TCR) would result in decreased transcription of IL-2

One of the TCR components, the CD3 chain, is significantly decreased in SLE T cells and in its place is the closely related common γ chain of the immunoglobulin receptors (FcR), a molecule not normally expressed in T cells

This substitution results in increased TCR responsiveness

The molecular mechanism of the IL-2 defect has now been clarified

The decreased IL-2 production observed in SLE patients is based on the relationship between the transcription factors CRE-binding protein (CREB) and CRE-modulator (CREM)

CREB occupies the binding site in resting T cells and upon activation, it is phosphorylated (pCREB) thereby promoting IL-2 transcription

IL-2 transcription is repressed by the replacement of pCREB by phosphorylated CREM (pCREM)

SLE patients have higher levels of CREM than CREB, resulting in decreased IL-2 production

Additionally, SLE T cells have increased levels of the serine/threonine phosphatase PP2A, the phosphatase primarily responsible for the dephosphorylation of CREB, and this contributes to decreased IL-2 production

BIOINFORMATICS TOOL PolyPhen-2 (Polymorphism Phenotyping v2) is a tool which predicts

possible impact of an amino acid substitution on the structure and function of a human protein using straight forward physical and comparative considerations.

PROVEAN (Protein Variation Effect Analyzer) is a software tool which predicts whether an amino acid substitution or has an impact on the biological function of a protein.

PROVEAN is useful for filtering sequence variants to identify non synonymous or variants that are predicted to be functionally important.

A fast computation approach to obtain pairwise sequence alignment scores enabled the generation of precomputed PROVEAN predictions for 20 single AA substitutions and a single AA deletion at every amino acid position of all protein sequences in human and mouse.

Swiss-PdbViewer or DeepView is an application that provides a user friendly interface allowing to analyze several proteins at the same time. The proteins can be superimposed in order to deduce structural alignments and compare their active sites or any other relevant parts

Uniprot is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequence and functional information

SWISS-MODEL is a fully automated protein structure homology-modelling server, accessible via the ExPASy web server, or from the program DeepView (Swiss Pdb-Viewer). The purpose of this server is to make Protein Modelling accessible to all biochemists and molecular biologists worldwide.

A Ramachandran plot (also known as a Ramachandrandiagram or a [φ,ψ] plot), originally developed in 1963 by G. N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan, is a way to visualize energetically allowed regions for backbone dihedral angles ψ against φ of amino acid residues in protein structure

PyMOL is an open-source, user-sponsored, molecular visualization system created by Warren Lyford DeLano 

PyMOL can produce high-quality 3D images of small molecules and biological macromolecules, such as proteins

The PSIPRED Protein Sequence Analysis Workbench aggregates several UCL structure prediction methods into one location. Users can submit a protein sequence, perform the predictions of their choice and receive the results of the prediction via e-mail or the web.

SOPMA SECONDARY STRUCTURE PREDICTION METHOD

IL2: SYSTEMIC LUPUS ERYTHEMATOSUS DISEASE; No. of SNPs (Homo sapiens) = 410; No. of non-synonymous SNPs = 36 for Human

Selected rs id on the basis of the cutoff value and predictionFor PolyPhen- (≥1 cutoff range , Probably damaging )For PROVEAN- (<-2.5 cutoff range , Deleterious)

IL2: SYSTEMIC LUPUS ERYTHEMATOSUS DISEASE; No. of SNPs (Homo sapiens) = 410; No. of non-synonymous SNPs = 36 for Human

FASTA SEQUENCE 1. Native SLE >sp|P60568|IL2_HUMAN Interleukin-2 OS=Homo sapiens GN=IL2 PE=1

SV=1MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

2. rs386556267 Pro22Leu >sp|P60568|IL2_HUMAN Interleukin-2 OS=Homo sapiens GN=IL2 PE=1

SV=1MYRMQLLSCIALSLALVTNSALTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT  

3. rs759849798 Leu73Pro >sp|P60568|IL2_HUMAN Interleukin-2 OS=Homo sapiens GN=IL2 PE=1

SV=1MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEPKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT  

4. rs778851120 Thr121Ile   >sp|P60568|IL2_HUMAN Interleukin-2 OS=Homo sapiens GN=IL2 PE=1

SV=1MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSEITFMCEYADETATIVEFLNRWITFCQSIISTLT  

Leu73Pro

Thr121Ile

Pro22Leu

NATIVE MUTANT

1MUTANT

2MUTANT

3

HOMOLOGY MODELLING USING PYMOL VIEWER

SEQUENCE IDENTITY

SEQUENCE SIMILARITY

SEQUENCE COVERAGE

NATIVE IL2 100.00% 0.60 0.87MUTANT IL2_1 99.25% 0.59 0.87MUTANT IL2_2 99.25% 0.59 0.87MUTANT IL2_3 99.25% 0.59 0.87

SWISS PDB VIEWER RESULTS

RAMACHANDRAN PLOT OF NATIVE AND MUTANT 1

NATIVE MUTANT 1

RAMACHANDRAN PLOT OF MUTANT 2 AND MUTANT 3

The Ramachandran plot  is the 2d plot of the φ-ψ torsion angles of the protein backbone. It provides a simple view of the conformation of a protein. The φ-ψ angles cluster into distinct regions in the Ramachandran plot where each region

corresponds to a particular secondary structure.

MUTANT 2 MUTANT 3

NATIVE IL2

MUTANT 1

Secondary Structure Prediction (PSIPRED)

MUTANT 2

MUTANT 3

Alpha helix Beta turn Random coil Length

Native IL2 83 is 54.25% 10 is 6.54% 36 is 23.53% 153

MUTANT IL2_1 87 is 56.86% 10 is 6.54% 35 is 22.88% 153

MUTANT IL2_2 79 is 51.63% 10 is 6.54% 40 is 26.14% 153

MUTANT IL2_3 83 is 54.25% 10 is 6.54% 35 is 22.88% 153

SOPMA SECONDARY STRUCTURE PREDICTION RESULT

CONCLUSION IL-2 is important for regulating T cell immunity and therefore

affects many aspects of immune responses

Not only is IL-2 dysregulated in SLE, but the IL-2 family members IL-15 and IL-21 are also expressed aberrantly

The decreased production of IL-2 in SLE patients most likely contributes to various immune defects such as decreased  production, decreased AICD, and potentially decreased CTL responses

A potential decrease in CTL activity is of utmost importance in SLE as decreased cytotoxicity would make patients more susceptible to intracellular infection

There is evidence that SLE patients have a higher incidence of Epstein Barr virus (EBV) infection and it has been reported that SLE patients have forty times higher viral load and a greater repertoire of antigen-specific B cells for EBV viral proteins

IL-2 deficiency affects multiple regulatory pathways in the host and in the case of SLE this contributes to the multifaceted dysregulation of the immune response

From the results obtained by computational algorithms it is conclude that SNPs with rs ids rs386556267, rs759849798, and rs778851120 were found to be probably damaging by PolyPhen tool with PSIC score was 1 for each and deleterious by PROVEAN tool