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Protein evolution The role of domains
Alice Skoumalová
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• Definition of a domain
• an independent structural, functional and
  evolutionary unit
• Structural unit
• Self-stabilizing locally folded region of
  tertiary structure
• Combination of motifs a-helix and ß-sheet
• Most proteins have 2 and more domains
• 2. Functional unit
• Various functions
• Ligands binding, membrane transit, catalytic
  activity,
• DNA binding, protein-protein interaction, etc.
• An independent function, cooperation with other
  domains
• 3. Evolutionary unit
• The relationship of proteins(superfamilies
  formation)
• The family tree
• SCOP (Structural Classification of Proteins)
  with 1200 protein superfamilies

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• The creation of new proteins
• duplication, divergence and recombination of
  domains
• new function (sequence divergence, combining with
  other domains)
• This mechanism facilitates the creation of
  proteins from different protein domains (no need
  of new genes for the formation of new proteins)

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• The recombination of domains
• Two generic principles

A domain can perform the same function, but in
different protein contexts (with different
partner domains) Syntactical shift
A domain can diverge and aquire a novel or
modified function Semantic shift
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Transcription factor FadR
Human methionine aminopeptidase
WHD
Oligomerisation/CoA-binding domain
Creatinase/aminopeptidase domain
Restriction endonuclease Fokl
WHD (Winged helix domain)
WHD
Catalytic domain
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• The creation of new proteins
• by the domain recombination (an example)
• Proteins that participate in the haemostasis
• form the superfamily of the related proteins
  (duplication, reception or deletion of the
  specific domains)
• contain a domain that is homologous to trypsin
  (they have a common ancestor with trypsin)
• the family tree can be generated with 7 gene
  modules

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Ancestral protein Trypsin-like serine protease
P
A modul which codes the structure called kringle
Kringle addition
P
K
Parent of all proteins
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P
EGF domain addition
K
P
Urokinase
K
P
E
Fibronectin domain 2 addition
K
P
E
F2
Fibronectin domain 1 addition
Kringle duplication
K
P
E
F2
F1
K
K
P
E
F2
EGF domain duplication
t-PA
K
P
E
E
F2
F1
Factor XII
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P
K
P
Propeptide addition
P
K
K
P
E
Urokinase
Pr
Calcium binding domain addition
K
P
E
F2
P
K
C
Pr
Kringle duplication
Kringle deletion
P
Pr
C
K
P
E
F2
F1
K
K
C
P
2 EGF domains addition
Pr
K
K
P
E
F2
Prothrombin
t-PA
C
E
E
P
Pr
Factors VII, IX, X Protein C
K
P
E
E
F2
F1
Factor XII
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P
K
P
Repeat kringle duplication
Urokinase
P
K
K
P
E
Pr
Hepatocyte growth factor
K
P
E
F2
K
K
K
K
P
P
K
C
Pr
Kringle duplication
K
K
K
K
K
P
P
Pr
C
K
P
E
F2
F1
Plasminogen
K
K
C
P
Pr
K
K
P
E
F2
Prothrombin
K
K
K
K
K
P
K
t-PA
C
E
E
P
Pr
Apolipoprotein (a) Constits of 40 kringles
Factors VII, IX, X Protein C
K
P
E
E
F2
F1
Factor XII
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• From the example above we can deduce
• The relationship of the haemostatic proteins is
  an example of the universal principle of the new
  protein creation
• Simple arithmetic operations with gene modules
  facilitate the creation of new proteins with
  different properties

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• Summary
• There is no simple relation 1 gene - 1 protein
• One gene can produces more proteins (various
  conformations, various domain recombination)
• Duplication, divergence and recombination of
  domains are crucial for the protein creation
  (there si no need of new genes for the new
  proteins formation)
• An example of relationship of proteins
  participating in the haemostasis

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Proteomics
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• What is proteomics?
• The large-scale study of proteins

Proteomics
Genomics
PROTEingenOME
Expression
Genom
Proteom
posttranslational modification alternative
splicing alternative folding
All proteins produced by an organism The human
body contains millions proteins One organism has
different protein expression in different parts
of its body, stages of its life cycle and
environmental conditions
All genes in DNA of an organism The human genome
contains 20-25000 genes The genom is a constant
entity
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• Increase in protein diversity
• Posttranslational modification
• Alternative splicing
• Alternative folding
• Primary transcript
• mRNA before the posttranscriptional modification

Alternative splicing
Posttranslational modification
Alternative folding
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• Posttranslational modification
• The chemical modification of a protein after its
  translation
• Addition of functional groups (acetate,
  phosphate, lipids, carbohydrates)
• Modification of amino acids
• Structural changes ( the formation of disulfide
  bridges, proteolytic cleavage)

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Alternative splicing of a pre-mRNA transcribed
from one gene can lead to different mature mRNA
molecules and therefore to different proteins
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Alternative folding The protein folding proceeds
from a disordered state to progressively more
ordered conformations corresponding to lower
energy levels
Local minimum (alternative conformation)
Global minimum (native state)
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Basic proteomic analysis scheme
Protein mixture
1. Separation 2D-PAGE
Individual proteins
2. Spot cutting Trypsin digestion
Peptides
3. Mass analysis Mass spectroscopy
4. Sequence analysis Peptide fragmentation
Peptide mass
Sequence information
5. Database search
Protein identification
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2D gel electrophoresis The synchronous analysis
of hundreds or even thousands of
proteins Proteins spread out on the surface
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• Application of proteomics in medicine (disease
  proteomics)

The role of proteins in the pathogenesis of
diseases
Protein expression in diseases
Using specific protein biomarkers to diagnose
disease Alzheimer disease (amyloid ß) Heart
disease (interleukin-6 and 8, serum amyloid A,
fibrinogen, troponins) Renal cell carcinoma
(carbonic anhydrase IX)
Biomarkers of diseases
Design of new drugs
Information about proteins causing diseases is
used for the identification of potential new drugs
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Biomarkers of diseases

• Proteome-based plasma biomarkers for AD
• Diagnosis of AD
• On clinical groundspost mortem (histology)
• There is no reliable diagnostic test
• Plasma may offer a rich source of disease
  biomarkers

• Identification of diagnostic biomarkers in the
  blood by proteomics
• Plasma samples of patients and control were
  analysed by 2D gel electrophoresis
• Spots that were significantly different between
  case and control groups were excised and analysed
  by mass spectroscopy

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• Results
• 15 spots were significantly different between
  patients and controls
• MS analysis ?2-macroglobulin, complement factor
  H,

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Virtual ligand screening
The identification of new drugs to target and
inactivate the HIV-1 protease (cleaves a very
large HIV protein into smaller, functional
proteins virus cannot survive without this
enzyme it is one of the most effective protein
targets for killing HIV)
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• Summary
• Proteomics studies proteins, particularly their
  structure, function and interaction
• The genome has already been analysed, now
  scientists are interested in the human proteome
  (millions of proteins)
• Key technologies used in proteomics are 2D gel
  electrophoresis and mass spectrometry
• Proteins play a central role in the life of an
  organism, their malfunction startes diseases
  proteomics is instrumental in discovery of
  pathogenesis of disease, biomarkers and
  potential therapetic agents

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Questions

• Definition of a domain (3 aspects), mechanisms of
  the new protein creation (in general),
  syntactical and semantic shift (the principle)
• Increase in protein diversity compared to genom
• Identification of the renal carcinoma biomarkers
  in the plasma
• Using of computer sofware for the development of
  new drugs