Evolution of paralogous proteins

1
Evolution of paralogous proteins

• Level 3 Molecular Evolution and Bioinformatics
• Jim Provan

Patthy Sections 7.1 - 7.3
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Advantagous duplications

• Duplication of a complete protein-coding gene
  results in two identical duplicons
• Encode same protein and express it in the same
  way as original
• Duplication will be advantageous if increased
  supply of gene product is advantageous (e.g
  histones or ribosomal proteins)
• Duplication by retroposition results in a
  processed duplicon
• Same protein encoded but expression likely to be
  different
• Duplication will be advantageous if change in
  expression pattern (tissue specificity,
  developmental stage) has biological advantage
• Fully redundant functions (neutral duplications)
  may be lost through drift but may persist long
  enough to acquire an advantageous function
• New proteins generally arise through modulation
  of existing ones

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Advantageous duplication of unprocessed genes

• Duplications are advantageous and maintained by
  positive selection if having multiple genes
  performing the same function ensures enhanced
  efficiency e.g. histones
• Positive selection for advantageous gene
  duplications can be observed in
• Insects exposed to insecticides
• Tumours and protozoa exposed to drugs
• Various stomach lysozymes in ruminants
• Seven closely related mRNAs encoding lysozymes
• Multiple lysozyme genes arose through duplication
• Serial duplication of lysozyme gene was an
  adaptive response to increase the expression of
  lysozyme

4
Advantageous duplication of processed genes

• Due to changes in chromosomal environment and/or
  5 regulatory regions, processed gene may have
  altered regulatory features and may not be
  competing with its progenitor
• Lower fidelity of reverse transcription means
  that duplicon may have deleterious mutations and
  be lost
• Two examples of positive selection increasing the
  chance of survival of testis-specific processed
  genes
• Phosphoglycerate kinase retrogene
• Pyruvate dehydrrogenase E1a retrogene

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The phosphoglycerate kinase retrogene

• Two functional PGK loci in the mammalian genome
• PGK-1 is X-linked and expressed constitutively in
  all somatic cells
• PGK-2 is a functional autosomal gene expressed in
  a tissue-specific manner exclusively in the late
  stages of spermatogenesis
• PGK-2 lacks introns and has a poly(A) tail
  processed gene
• Evolution of PGK-2 was a compensatory response to
  inactivation of the X-linked gene before meiosis
• Mature spermatozoa require PGK to metabolise
  fructose in semen
• X-inactivation called for a functional autosomal
  PGK locus
• Unequal crossing-over would not have solved this
  problem
• Processed gene had an initial advantage since it
  permitted the expression of PGK in a tissue where
  the X-linked gene was inactivated
• Subsequently evolved a testis-specific promoter

6
The pyruvate dehydrogenase E1a retrogene

• PDH E1a subunit of the PDH complex is located on
  the X chromosome and is expressed in somatic
  tissues
• Another PDH E1a locus is found on chromosome 4
• Testis-specific and expressed in postmeiotic
  spermatogenic cells
• Lacks introns, has a downstream poly(A) tract and
  is flanked by a pair of 10 bp direct repeats
• After the last meiotic division, spermatids rely
  on energy from pyruvate for the maturation
  process PDH E1a is essential
• X-chromosome is inactivated in postmeiotic
  spermatogenic cells
• Only half the cells contain an X-chromosome
• Evolution of an alternative, non-X-linked gene

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Neutral duplications

• If there is no selective advantage from a
  duplication event, functional constraints
  protecting the new gene from deleterious
  mutations may be relaxed
• May ultimately be converted to a pseudogene
• Many clusters of duplicated genes contain
  pseudogenes
• Advantageous mutations, either in the coding
  sequence or the regulatory region, may lead to
  positive selection
• Where there is a major change of function,
  several critical sites may be involved
• New function might not be fully manifested until
  several sites have adapted
• Early mutational steps may be selectively neutral

8
Visual pigment proteins

• Old World primates have three colour-sensitive
  proteins
• Green- and red-absorbing photoreceptors are
  encoded by a pair of closely related (96
  identity), closely linked genes on the X
  chromosome - suggests very recent gene
  duplication
• Blue photoreceptor is encoded by an autosomal
  gene
• New World monkeys have only one X-linked pigment
• Duplication must have occurred in the ancestor of
  Old World monkeys after divergence from New World
  monkeys
• Humans, apes and Old World monkeys can
  discriminate three colours whereas New World
  monkeys can only distinguish two
• Prior to emergence of three pigments, rate of
  non-synonymous substitution exceeded synonymous
  rate suggests positive selection for
  three-colour vision

9
Serine proteinases and their inhibitors

• Pancreatic proteinases (trypsin, chymotrypsin and
  elastase) illustrate paralogues with minor
  modifications of function
• Strikingly similar three-dimensional structures
• Very different substrate specificities
• Elastase cleaves residues with small, non-polar
  side chains (Ala, Val etc.)
• Chymotripsin cleaves at bulky, hydrophobic
  residues (Phe-X, Tyr-X etc.)
• Trypsin cleaves only Arg-X or Lys-X
• Advantage of having multiple digestive
  proteinases is clear their combined activities
  ensure more efficient utilisation of proteins in
  foodstuffs.
• Original duplicons survived since they acquired
  advantageous mutations that diversified their
  function

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Serine proteinases and their inhibitors

• Molecular basis of differences in substrate
  specificity can be rationalised from
  three-dimensional structure and understanding of
  the catalytic mechanism
• Specificity of trypsins for Arg or Lys residues
  is due to
• Deep substrate binding site which can accommodate
  side chains
• Asp-189 residue at the bottom of the pocket which
  neutralises the Arg / Lys residue of the
  substrate
• Specificity of chymotrypsin for bulky aromatic
  residues due to
• Large, hydrophobic substrate binding pocket
• Small, neutral (usually Ser) residue at position
  189
• Elastase has shallower binding site
• Residues 216 and 226 have small side chains in
  trypsin/chymotrypsin
• Elastase has bulkier residues (Val, Thr) at these
  positions

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Serine proteinases and their inhibitors

• Substitution of a few, key residues can alter
  sequence specificity without eliminating enzyme
  activity
• Replacement of Asp-189 of trypsin with a Ser
  residue (to mimic chymotrypsin) greatlydiminishes
  activity towards Lys or Arg and increases
  specificity for hydrophobic substrates 10- to
  50-fold
• Lack of complete change of substrate suggests
  that other readjustments had to occur during
  divergence of trypsin and chymotrypsin from their
  common ancestor
• Supports notion that new function may emerge by
  continual improvement of function
• Correlated with functional adaptation of serine
  proteinase inhibitors
• Porcine elafin genes have 93-98 conservation in
  introns but only 60-77 similarity in exon 2,
  which encodes the inhibitor domain
• Due to accelerated mutation rate KA gtgt KS

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UDP-glucuronosyltransferases (UDPGTs)

• UDPGTs detoxify hundreds of compounds by
  conjugation and increasing water solubility to
  facilitate excretion
• In mammals, bilirubin (by-product of haem
  turnover) must undergo detoxification by
  conjugation to glucuronic acid
• Glucuronidation is carried out by a large family
  of UDPGTs with different, but overlapping,
  substrate specificities
• Means that UDPGTs also affect levels of several
  hormones
• Overproduction (through whole-gene duplication)
  deleterious
• UDPGTs have distinct domains serving different
  functions
• N-terminal globular domain which binds toxic
  substrate
• C-terminal globular domain involved in
  UDP-glucuronic acid binding
• Substitutions in restricted regions of N-terminal
  domain led to diversification of substrate
  specificities

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UDP-glucuronosyltransferases (UDPGTs)

• Two families of UDPGTs in mammals which differ
  markedly in evolutionary strategy used for
  functional diversification
• UDPGT2B subfamily has evolved through classic
  process of whole gene duplication resulting in
  several isoforms
• Clustered gene family on chromosome 4
• Primary substrates include 4-hydroxysterone and
  hyodeoxycholic acid
• Single UDPTG1 gene complex on chromosome 2 has
  diversified by duplication only of exon 1, which
  encodes substrate-binding domain
• Human UDPGT1 gene complex has six closely related
  exon 1 variants
• Single set of four exons that encode the
  C-terminal parts of UDPGTs
• mRNAs of different isoforms produced by
  differential splicing of one of the exon 1
  variants onto the constant C-terminal exons

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Major change of function in paralogous genes

• Some members of the serine protease family (e.g.
  haptoglobin, hepatocyte growth factor,
  azurocidins) have lost their capacity to act as
  proteinases
• Have lost one or more of the residues in the
  catalytic triad
• Have other important biological functions
• Haptoglobin binds globin release from lysed
  erythrocytes
• Hepatocyte growth factor acts through specific
  receptor tyrosine kinases to stimulate cell
  growth
• Azurocidin has bactericidal activity
• Careful analysis sometimes indicates plausible
  pathway for transition from one function to
  another

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Evolution of azurocidins

• Azurophil granules of neutrophils contain several
  proteins implicated in the killing of
  microorganisms
• Serine proteases that cause degradation of
  connective tissues (cathepsin G, neutrophil
  elastase, proteinase 3)
• Azurocidin is similar to these but lacks His-57
  and Ser-195 and thus has no proteolytic activity
• Bactericidal activity of azurocidin mediated by
  tight binding to anionic lipopolysaccharide, a
  component of the Gram-negative bacterial
  envelope
• Serine proteinase fold used as a scaffold for
  endotoxin binding
• Fact that azurocidins share most recent common
  ancestor with proteinases that have antibacterial
  activity suggests that this was a common function
  of the ancestor
• New function probably emerged before original
  function was lost

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Major change of function by domain acquisition

• In proteinases involved in blood coagulation,
  very large segments are joined to the
  trypsin-homologue region
• These nonproteinase parts of plasma proteinases
  consist of multiple structural-functional domains
  that were introduced by exon shuffling
• Function modified not only by point mutations but
  also by domain insertions and duplications
• Proteinase domains retained proteolytic activity
  but point mutations led to a altered (usually
  narrower) sequence specificity
• Value of domain-acquisition mutations is that
  they can endow novel binding specificities and
  lead to dramatic changes in regulation and
  targeting

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Modular structure of blood coagulation and
fibrinolytic proteinases
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Domain acquisition in the evolution of plasma
proteinases

• Selective value of domains joined to proteinase
  domain illustrated by fact that they are usually
  involved in interactions with cofactors,
  substrates or inhibitors
• Vitamin K-dependent calcium-binding domains of
  prothrombin, coagulation factors VII, IX, X and
  protein C anchor proteinases to phospholipid
  membranes ensuring proper regulation of cascade
• Kringle domains of plasmin and plasminogen are
  critical for binding of proteinase to its primary
  substrate, fibrin
• Serine proteinase domain has proteinase
  specificity very similar to that of trypsin
  (Lys-X and Arg-X)
• Fibrin specificitydue to fact that kringle
  domains have specific fibrin-binding sites that
  target the enzyme to fibrin

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Similarities and differences in the evolution of
paralogous and orthologous proteins

• Common protein folds are conserved in both
  paralogues and orthologues and structural
  elements generally accept mutations at similar
  rates between the two
• One difference is that orthologous proteins are
  likely to fulfil very similar functions in
  different species whereas paralogous proteins are
  more likely to have diversified in function
• When comparing orthologous proteins, residues
  that are critical for structure, function and
  specificity are equally likely to be conserved
• When comparing paralogous proteins that fulfil
  different functions, only residues essential for
  structure are likely to be conserved