Transcript PAINT-Apoptosis-summary-Pascalex

PAINT apoptosis annotation
Lessons leaned
Apoptotic process definition
A programmed cell death process which begins when a cell receives
an internal (e.g. DNA damage) or external signal (e.g. an
extracellular death ligand), and proceeds through a series of
biochemical events (signaling pathways) which typically lead to
rounding-up of the cell, retraction of pseudopodes, reduction of
cellular volume (pyknosis), chromatin condensation, nuclear
fragmentation (karyorrhexis), plasma membrane blebbing and
fragmentation of the cell into apoptotic bodies. The process ends
when the cell has died.
The process is divided into a signaling pathway phase, and an
execution phase, which is triggered by the former.
Execution phase of apoptosis
A stage of the apoptotic process that starts with the controlled
breakdown of the cell through the action of effector caspases or
other effector molecules (e.g. cathepsins, calpains etc.). Key steps
of the execution phase are rounding-up of the cell, retraction of
pseudopodes, reduction of cellular volume (pyknosis), chromatin
condensation, nuclear fragmentation (karyorrhexis), plasma
membrane blebbing and fragmentation of the cell into apoptotic
bodies. The execution phase ends when the cell has died.
Execution phase of apoptosis
• Well defined, clear beginning and end
• Based on GO annotation guidelines, expect
proteins annotated to this term to participate
in the execution phase of apoptosis
• PAINTers annotated families containing
human proteins annotated to ‘execution
phase of apoptosis’ or children
“Execution of apoptosis” and children
PAINT apoptosis - summary
• 52 families annotated:
- 8 were participants in execution phase of
apoptosis; 44 others are either:
(a) upstream of apoptosis
(b) targets
(c) phenotypes
Example 1: Protein upstream of
apoptosis execution
Cytochrome c annotated to apoptotic DNA fragmentation
Fig 7C HeLa cell S-100 depleted of cytochrome c
using the monoclonal anti-cytochrome c
antibody lost the dATP-dependent activation of
CPP32 and the ability to induce DNA
fragmentation
in
the
added
nuclei
PMID: 8689682
 [Cells] – [cytochrome c] = No caspase 3 (CPP32) activation; No DNA fragmentation
 [Cells] – [cytochrome c] + [cytochrome c] = Caspase 3 activation; DNA fragmentation
What else do we know ?
 Apoptotic stimulation release cytochrome
c into the cytoplasm
 Cytochrome c is required for cleavage of
caspase 3
 Note that the authors do not claim that this
is a direct effect: “Immunodepletion of
cytochrome c from these cytosols resulted in
the loss of CPP32 activating activity “
Paper findings
Apoptotic stimulus
cytochrome c release *occurs after*
apoptotic stimulation
Cytochrome c release from mitochondria into cytosol
cytochrome c is *required for* CASP3
activation and DNA fragmentation
Caspase 3 activation
DNA fragmentation
What can we infer ?
1. cytochrome c is a participant in apoptosis because its biological activity occurs after
the inducing stimulus [start]
2. cytochrome c is upstream of the apoptotic DNA fragmentation and caspase activation
Example 2: Phenotype of overexpression
• E3 ubiquitin-protein ligase TRAF7 Q6Q0C0
annotated to execution phase of apoptosis
 Exogenous expression of TRAF7
reduces cell survival and
increases DNA fragmentation.
 No other data in terms of where
in apoptosis this may be. All we
know is altering TRAF7 levels
affects apoptosis.
Example 3: Target
DSG2 was annotated to execution phase of apoptosis
DSG2 is a *target* of a protease (caspase), and although its degradation indeed
seems to be a part of apoptosis it does not *mediate* apoptosis.
Problem with targets may result from
inconsistent guidelines
• Guideline 1: Annotate all proteins that have a
function during a process, ie between the beginning
and the end
• Guideline 2: Do not annotate targets of processes
Guidelines are consistent if we only annotate
*active participants* in processes
For example: kinase and phosphorylation: the target of a kinase does not mediate
phosphorylation or to the target of the degradation that would not be annotated to
this process of "protein catabolic process"
Capturing targets
 Annotation extension may resolve that, but only
in cases where we know which protease cleaves
the protein.
LEGO: [GO:NEW] PROTEASE ACTIVITY INVOLVED IN cellular component disassembly
involved in execution phase of apoptosis
ENABLED_BY unknown_protein
TARGET DSG2
until then… ???
Why does this matter ?
• How can users find out what proteins are
involved in a specific process ?
Survey of the annotations
to ‘execution of apoptosis’ and children by EXP (partial list)
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Inhibitor of apoptosis PTHR10044
Calpain
PTHR10183
Caspase
PTHR10454
Sorting nexin
PTHR10555
Ubiquilin PTHR10677
Protein arginine n-methyltransferase PTHR11006
Peptidyl-prolyl cis-trans isomerase PTHR11071
Msf1/px19 related
PTHR11158
Palmitoyl-protein thioesterase/dolichyldiphosphatase 1 PTHR11247
Bcl-2 related
PTHR11256
Ephrin PTHR11304
Deoxyribonuclease
PTHR11371
Glucagon PTHR11418
Type I interferon PTHR11691
30s/40s ribosomal protein s3PTHR11760
40s ribosomal protein s3a PTHR11830
Ets (transcription factor)
PTHR11849
Survey
of
the
annotations
to ‘execution of apoptosis’ and children by EXP (partial list)
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Ribonuclease ph related PTHR11953
Cytochrome c PTHR11961
Group ii pyridoxal-5-phosphate decarboxylase
PTHR11999
Bcl2/adenovirus e1b 19-kda protein-interacting protein PTHR12112
Polymyositis/scleroderma autoantigen-related
PTHR12124
Cell death activator cide PTHR12306
Rho-type gtpase activating protein
PTHR12659
U3 small nucleolar rna-associated protein 11 PTHR12838
Map-kinase activating death domain protein (madd)/denn/aex-3(c.elegans) PTHR13008
Dna fragmentation subunit beta PTHR13067
Microtubule-associated protein PTHR13843
Endonuclease related
PTHR13966
Helicase-related
PTHR14950
Family not named (bnip3) PTHR15186
Cop9 signalosome complex subunit 7/dendritic cell protein ga17 PTHR15350
Rbm25 protein
PTHR18806
Family not named (steroid receptor rna activator 1 ) PTHR18834
Protein disulfide isomerase
PTHR18929
Tumor protein d52 PTHR19307
Survey of the annotations
to ‘execution of apoptosis’ and children by EXP (partial list)
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Telomeric repeat binding protein PTHR21717
Disco-interacting protein 2 (dip2)-related
PTHR22754
F-box and wd40 domain protein PTHR22844
Family not named PTHR22845
Disulfide oxidoreductase PTHR22912
Nadh dehydrogenase-related
PTHR22915
Myosin light chain kinase-relatedPTHR22964
3'-5' exonuclease eri1-related PTHR23044
Paraneoplastic antigen
PTHR23095
Translation factor PTHR23115
Family not named (desmocollin - cadherin family - plasma membrane/adhesion protein)
Family not named (granzyme) PTHR24271
Family not named PTHR24272
Family not named (ptgis) PTHR24306
Akt-related serine/threonine-protein kinase PTHR24352
mitogen-activated kinase kinase kinase PTHR24361
PTHR24025
Regulatory roles versus indirect roles
prote
ase
DFFA
DFFB
DFFA bound to DFFB inhibits DFFB’s nuclease activity
DFFA
DFFB
Apoptotic stimulus results in degradation of DFFA
DFFB = Deoxyribonuclease activity and Apoptotic DNA fragmentation
DFFA = Regulation of apoptotic DNA fragmentation
no MF (need something like nuclease inhibitor activity)
Regulation:
Regulation of apoptotic DNA fragmentation
• Used when ‘directness’ of the role of a protein in a process is not
clear; especially with mutants
• ‘True’ regulation cannot be captured explicitly
Gene/Product
Aifm1
Apaf1
BAX
Casp3
CDKN2A
DFFA
PRELID1
Gene/Product name
apoptosis-inducing factor, mitochondrion-associated 1
apoptotic peptidase activating factor 1
Apoptosis regulator BAX
caspase 3
Cyclin-dependent kinase inhibitor 2A, isoform 4
DNA fragmentation factor subunit alpha
PRELI domain-containing protein 1, mitochondrial
Evidence
IGI
IGI
IMP
IMP
IMP
IDA
IDA
Why does this matter ?
• The usefulness of the more granular terms is
lost; this amounts to having everything
annotated to ‘apoptotic process’
 Efforts on ontology development and complexity of annotation are wasted
• What’s the impact on analysis tools that use GO ?
How widespread is this in the GO ?
• Particular issue for apoptosis is that many
‘processes’ are also experimental read-outs
Apoptosis assays
PMID: 12533214
GO terms corresponding to
apoptosis assays read-outs (1/2)
Assay
GO term
Nuclear and cytoplasmic • GO:0006921 cellular component disassembly involved in
condensation
execution phase of apoptosis
• GO:0030263 apoptotic chromosome condensation
DNA fragmentation
• GO:0006309 apoptotic DNA fragmentation
• GO:0043653 mitochondrial fragmentation involved in
apoptotic process
Caspase activity assay
• GO:2001270
regulation of cysteine-type endopeptidase
activity involved in execution phase of apoptosis
• GO:0006919
activation of cysteine-type endopeptidase
activity involved in apoptotic process
- Caspase 3 activity
assay
• GO:0097297 activation of cysteine-type endopeptidase
activity involved in execution phase of apoptosis
GO terms corresponding to
apoptosis assays read-outs (2/2)
Assay
GO term
Phagocytosis and autolysis
• GO:0043652 engulfment of apoptotic cell
• GO:1901074 regulation of engulfment of apoptotic
cell
Cytochrome C release
• GO:0001836
mitochondria
release of cytochrome c from
• GO:0090199
regulation of release of cytochrome c
from mitochondria
Phosphatidylserine
exposure on outer
membrane
• GO:0070782
phosphatidylserine exposure on
apoptotic cell surface
Open question
• What if we don’t know if a protein participates
in a process ?
• For processes, usually all we know is that the
protein acts upstream from the readout
measured
Take home message
• Beware of participation in process versus
experimental read-out
• Beware of granular terms: annotate only if
there is evidence
Semantics of a GO annotation
• What does it mean to associate a GO term to a gene product ?
• Typical experiment: mutant x has increased/decreased DNA
fragmentation/caspase activation, etc. upon treatment with
apoptotic-inducing stimulus
• Gene A annotated to DNA fragmentation involved in apoptosis
• Manipulating Gene A results in a change in Process 1; ie, there
is a causal relation between Gene A and Process 1.
• Semantics of existing annotations: Gene A is most likely
upstream of, and possibly a participant in Process 1.
• Semantics of existing ‘regulates’ annotations: Gene A is
upstream of the process, may regulate, but is not a participant
in the process.
Proposal
• Right now all annotations are potentially
‘upstream up’, especially IMPs, and doubleespecially IMP+ regulation.
• Use a new qualifiers?
upstream_of and participant_in
Implementation
• Proposed rule: if a protein is annotated to a
MF that has a has_part relation to a BP, then
the protein can be considered as being
‘participant’ in the process
• Advantage: handles the ‘regulation’ issue