Recerca de selenoproteïnes en el genoma d’organimes eucariotes

Bioinformàtica

Didac SantesmassesPhD student

didac.santesmasses@crg.eu

Bioinformatics and genomics programmeRoderic Guigó's group

Centre for Genomic Regulation, Barcelona

GPx6 (selenoprotein)

GPx5 (cysteine homologue)

Genes in human chromosome 6

The only selenoprotein in chromosome 6

Dark Blue: coding

Structure of a selenoprotein gene

Selenoprotein families include selenoproteins and cysteine

homologues (= orthologues or paralogues)

Selenoproteins are generally misannotated

Percentages are computed by comparison Selenoprofiles-Ensembl annotations – see Mariotti and Guigó, 2010 - Bioinformatics.

Bioinformatics methods for selenoproteins

• De novo: Selenogeneid (Castellano et al. 2001)• Homology based approaches:

UGA / Sec or UGA / Cys alignments

(e.g. Kryukov et al. 2003)

- Selenoprofiles (Mariotti and Guigó 2010)- Seblastian (Mariotti et al. 2013)

• SECIS prediction: - SECISearch (Kryukov et al. 2003) - SECISearch3 (Mariotti et al. 2013) – explained later

Why selenocysteine?

• What is cysteine used for?

mainly for: Disulfide bonds

Inter or intramolecular, important for the folding of proteins, their stability, and in many cases necessary for catalysis.

Thioredoxins are a large class of proteins that perform redox reactions using thiol/disulfide switches (catalytic cysteines).

Why selenocysteine?

The term “Thioredoxin” generally designates small oxidoreductase proteins, constituing a pool of enzymes for anti-oxidant defense. The thioredoxin system includes these proteins and those operating on them, both using them as electron donors, or maintaining them reduced.

Plenty of other proteins possess a thioredoxin-like fold, relying on the same local structure that includes a thiol/disulfide switch, and performing redox functions.

Selenocysteine is found almost always replacing a catalytic cysteine in redox proteins. Many selenoproteins possess a thioredoxin-like fold. Some are involved in the thioredoxin system. Consistently, selenocysteine is more reactive than cysteine, and it is a better reductant.

Anyway, some selenoproteins possess totally unrelated functions (e.g. SelJ = eye crystallin, SelP = selenium storage and transport)

Examples:Thioredoxin Reductases (TR)

TrxS

S TrxSH

SHTR +NADP-

+NADPH+H+

• TR3: mitochondrial Found in all vertebrates

• TGR (TR2): can reduce glutathione disulfide. Contains a glutaredoxin (Grx) domain

Found only in tetrapodes

• TR1: cytosolic. Found in all vertebrates

3 paralogous genes are present for this family in human, all of which are selenoproteins. Sec is present as penultimate residue, and it is catalytic.

TR are the only responsible for the reduction of thioredoxins in cell.

Glutathione Peroxidases (GPx)

3 groups:GPx1/GPx2, GPx3/GPx5/GPx6, GPx4/GPx7/GPx8

GPx6 converted to Cys independently in 3 species

• GPx1: cytosolic, abundant in liver and red blood cells

• GPx2: cytosolic, found in liver and gastrointestinal system

• GPx3: secreted, found in plasma and intestine

• GPx4: cytolic / mytochondrial, abundant in testis. Can reduce phospholipid hydroperoxides

• GPx5: secreted/membrane bound.Found only in epididymis

• GPx6: secreted (?).Found in olfactory epithelium and embryonic tissues

• GPx7: secreted (?)

• GPx8: membrane bound (?)

R-OOH + 2 GSH R-OH + H2O + GSSGGPx

Reduces superoxides at expenses of glutathione (GSH).8 paralogues in human, 5 with Sec

28

20 duplications

9 gene losses

13 Sec Cys

Selenoproteins in mammals / vertebrates

Among vertebrates, selenoproteins are quite conserved: most of them are found in all species, few transformations occurred. This is very different than the situation in insects:

(Chapple and Guigó, 2008)

• Selenoproteins have the peculiar characteristic of possessing a UGA codon, recoded because of the presence of the SECIS element.

• If you learn how to predict selenoproteins, you are able to do the same with any “standard” protein family.

• Bioinformatics project: find all selenoproteins in a given genome

Selenoproteins as test case

UPF Biologia. Curs 2007-15

2007/08 – 2008/09: find all selenoproteins in a given protist genome2009/10 – 2011/12: find a given selenoprotein family in all protist genomes2012/13 – 2014/15: find all selenoproteins in a given vertebrate genome

Objective: find all selenoproteins in a given vertebrate genome

Project 2014-15 selenoproteins in vertebrates

http://bioinformatica.upf.edu/

Useful resourcesSequences of your assigned species:

• EnsemblCollection of genomes (and annotations)

Your assigned genomes will be available in the UPF

computers when you will start the project

Useful resourcesSequences of your assigned species:

• NCBI nucleotideCollection of all sequences (genomes, ESTs, etc)If you do not find something that you expect to be there, look for other

sources of sequences. Most genomes today are low/medium-quality

Useful resourcesSelenoprotein sequences:

• SelenoDB 2.0 (and 1.0)Database containing manual annotations for human, and

automated annotations (selenoprofiles) for other vertebrate species.

• NCBI proteinNCBI hosts the sets of all known proteins. Noisy, but

comprehensive. You can find here more selenoprotein sequences searching by homology (blast) or by keywords

Useful resources

Tools:• Blast - typically tblastn• Exonerate - protein2genome mode• Genewise

• Webserver with SECISearch3 and Seblastian:http://seblastian.crg.es/

S14. Anotació de genomes (I)

S15. Anotació de genomes (II)

Based on a manually curated 2ndary structure alignment

Combines up to 3 methods to ensure maximum sensitivity

Filter and grading procedure based on manual inspection of hundreds of SECIS elements

SECISearch 3

Mariotti et al. 2013

Assumptions: the presence of a detectable

SECIS within acceptable genomic distance from the Sec-

UGA annotated homologue(s) (Sec/Cys) in the reference

protein database

Seblastian

Mariotti et al. 2013

• Results must be presented in a web page with the structure of a scientific paper

• A file with the aminoacid sequences of all selenoproteins identified must be provided; plus another file with all SECISes identified

• All genes should be as complete as possible: starting with a AUG, ending with a stop codon, and with an identified SECIS element downstream

• Ignore alternative isoforms (if any), just choose one

Notes for the project

• Report also the genes of selenoprotein machinery: SecS, eEFsec, pstk, secp43, SBP2, SPS1, (SPS2).

Ignore tRNAsec, for technical reasons

Notes for the project

• Zero, one or many genes? Careful with superfamilies, and gene duplications

• Know what to expect

• Genome assemblies are not perfect!

Common pitfalls

EvaluationThe projects will be evaluated based on:

- results you are expected to find all selenoproteins in your assembly

- discussioninterpret your results logically

- methodsscripting is encouraged (but not compulsory)

- presentationthe web page should present the work as clearly as possible