Apoptosis Curation Manual

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Apoptosis curation manual (General guidelines for annotating to cell death-related terms)

Rationale and aim

The motivation behind the apoptosis GO project was originally to clean up and expand the node under GO:0006915 “apoptosis”, which contained poorly structured and connected terms that did not reflect the current scientific knowledge on such an important area of biomedical research. Once the project started, it became apparent that its scope needed broadening, and some cell death-related terms above apoptosis have also been edited. In the present document we aim to provide a curation guide, starting from the most general terms directly under “cell death”, and walking our way down the ontology, hopefully providing a “decision tree” for GO curators. In particular, this document is meant as a useful tool to possibly re-house annotations that are currently placed under very generic terms in the new ontology (i.e. “apoptotic process”, “regulation of apoptotic process” and “induction of apoptosis”). There are still many direct manual annotations to these broad terms (1591, 691 and 861 respectively), and a good proportion could be made more specific and therefore gain in information content. This draft does not aim to be fully exhaustive, and only the most relevant changes to the ontology made during the apoptosis project will be discussed. Regulatory terms are not discussed unless required. In several cases, we have added definition comments to cell-death related terms, as a ‘permanent’ guideline for curators. If you think that more terms would benefit from having such comments, or that their definitions are unclear, please let us know – your suggestions will be very welcome. The ontology work consisted in changes and additions to Biological Process (BP) terms under the “cell death” branch, and to Molecular Function (MF) terms that have a relationship with apoptosis (caspase-related terms). Because some of the changes and new definitions challenge and even contradict the old ontology, we highly recommend reading through this document before using the new terms. Now we will describe the most important branches of the cell death node as represented in the figure below (this is also provided as a separate file, cell_death_node_graph.docx).

Types of cell death other than apoptosis

Current scientific knowledge of different types of cell death is not as rich as it is for apoptosis, and it is sometimes difficult to ascertain that the process we want to curate is indeed a distinctive type of cell death. However, some are sufficiently characterized, and there are terms available for them to annotate with, as follows.

[GO:0070265] necrotic cell death and children

Contrary to apoptosis and other types of cell death, necrotic cell death is mainly characterized by the loss of integrity of the plasma membrane and the loss of intracellular contents. However, in some instances it can occur under controlled conditions. In this case, the process should be annotated to the new term “programmed necrotic cell death”, or to its more specific child “necroptosis”, as follows.

[GO:0097300] programmed necrotic cell death

This new term was required to update the placement of existing “necroptosis” (GO:0070266); it is a child of both “necrotic cell death” and “programmed cell death”. Its definition is the same as the old one for “necroptosis”: “A necrotic cell death process that results from the activation of endogenous cellular processes, such as signaling involving death domain receptors and Toll-like receptors.” The definition of “necroptosis” has been made more specific:

[GO:0070266] necroptosis

“A type of programmed necrotic cell death characterized by activation of receptor interaction protein kinase 1 and/or 3 (RIP1 and/or RIP3).” Necroptosis is the focus of several recent papers, and it can occur via formation of a protein complex called ripoptosome (see GO:0097342). Annotation example: See PMID:21052097, annotations to Birc2 (Q62210) and Birc3 (O08863).

[GO:0012501] programmed cell death and children

As discussed above, there are instances of programmed cell death that escape the definition of apoptosis. These may be annotated to the general term GO:0012501 “programmed cell death” or to one of its children. Also, the apoptotic process as defined in GO:0006915 does not occur in some taxa, such as plants. Where evidence is shown for a controlled (programmed) cell death modality in such species, the general term GO:0012501 “programmed cell death” may be used, and some specific children also exist: GO:0097468 “programmed cell death in response to reactive oxygen species” (with descendants GO:0010421 “hydrogen peroxide-mediated programmed cell death” and GO:0010343 “singlet oxygen-mediated programmed cell death”), and GO:0009626 “plant-type hypersensitive response” and its children, which are descendants of GO:0034050 “host programmed cell death induced by symbiont”.

Parthanatos, netosis, entosis, pyroptosis and other types of cell death

Although it has been proposed to create new, specific terms for each one of these subtypes of cell death, the discussions with the experts of the APO-SYS consortium made it clear that research in the field for most of these terms is still quite preliminary and it is not clear whether they reflect real biological events or not. For the time being, no new terms have been created, but the only existing one (GO:0070269 pyroptosis) has not been obsoleted because of an increasing number of publications on pyroptosis in the last few years.

[GO:0006915] apoptotic process

The term name has been changed from “apoptosis” to “apoptotic process”, to better reflect its composite nature. Two part_of children have been added to illustrate the division of this process in two distinctive phases (signaling and execution). This concept has been incorporated in the definition of GO:0006915. Direct annotations to ‘apoptotic process’ should be restricted to those instances where it is not possible to distinguish which phase is specifically involved. The phases and their children terms are discussed at the end of this section. As for direct is_a children terms of GO:0006915:

[GO:0043276] anoikis

This term indicates a specific type of apoptosis induced by inadequate adherence of the cell to its substrate.

[GO:0097285] cell-type specific apoptotic process and children

This term was created as a generic parent to group terms indicating occurrences of apoptotic process in specific cell types. The process of apoptosis may not vary, but the cell type does. Please do not annotate to the parent term, and refer instead to its more granular children, or to GO:0006915 “apoptotic process”.

Signaling and execution phases of apoptosis

The most important change in the apoptosis branch of the BP ontology is the creation of two part_of children of “apoptotic process”, to make a clear distinction between two phases: [GO:0097190] “apoptotic signaling pathway” and [GO:0097194] “execution phase of apoptosis”. During the signaling phase, only initiator / apical caspases (e.g., CASP2, CASP8, CASP9, and CASP10) are activated, and no breakdown of cellular structures occurs. The execution phase starts with the activation of effector / executive caspases (e.g., CASP3, CASP6, CASP7), and during its course cellular structures break down in a controlled fashion, ultimately leading to cell death and the formation of apoptotic bodies. The creation of parent terms depicting both phases allows for the correct classification of many children terms that describe more granular aspects of apoptosis.

[GO:0097190] apoptotic signaling pathway and children

Gene products involved in events that take place in order to activate initiator caspases can be annotated under this parent term or its corresponding regulatory terms. Most of the mitochondrial events related to apoptosis (discussed later) take place during this phase.

Extrinsic and intrinsic apoptosis

The term “apoptosis signaling pathway” has the children [GO:0097191] “extrinsic apoptotic signaling pathway” and [GO:0097193] “intrinsic apoptotic signaling pathway”. The existence of cross-talk points between these two pathways often makes it difficult to distinguish between them, as both extrinsic and intrinsic apoptosis can be mediated by mitochondrial events. To distinguish between these two pathways, curators should take the initial point of the signaling pathway as a reference, as detailed below. The more generic term ‘apoptotic signaling pathway’ can be used to annotate gene products involved in apoptotic events happening downstream of the cross-talk point between the extrinsic and intrinsic pathways. Coming from the extrinsic apoptotic signaling pathway, the cross-talk starts when caspase-8 cleaves Bid and the truncated product induces mitochondrial outer membrane permeabilization. From this point on it is not possible to distinguish between extrinsic and intrinsic pathways. Also, in instances where it is not known what kind of signal initiated the apoptotic process, the generic parent term ‘apoptotic signaling pathway’ should be used.

[GO:0097191] extrinsic apoptotic signaling pathway

It starts with the reception of an external signal at the level of the cell surface (either by ligand binding or by ligand withdrawal) and can be mediated by mitochondrial events or not. Children terms refer to specific signals that activate the pathway, the best-known case being [GO:0008625] extrinsic apoptotic signaling pathway via death domain receptors. It is important to note the new term [GO:0097192] extrinsic apoptotic signaling pathway in absence of ligand. This particular term refers to the apoptotic process triggered by the withdrawal of a ligand, such as a growth factor, that is under non-apoptotic conditions bound to a dependence receptor. It is important to remember that apoptosis triggered by growth factor withdrawal has been traditionally considered an instance of intrinsic apoptosis. However, the detailed characterization of the molecular mechanisms underlying this particular type of cell death resulted in a more appropriate classification as a specific variety of extrinsic apoptosis.

Annotation examples: [GO:0008625] extrinsic apoptotic signaling pathway via death domain receptors: See PMID:21525013, annotations to TWEAK (TNF12, O43508) and FN14 (TNFRSF12A, Q9NP84). [GO:0097192] extrinsic apoptotic signaling pathway in absence of ligand: See PMID:21172653, annotations to UNC5B (O08722) and PR65beta (PPP2R1B, P30154). A diagram of the extrinsic apoptotic pathway including examples of molecular players can be found in Figure 1 in PMID:21760595.

[GO:0097193] intrinsic apoptotic signaling pathway

It starts with the reception of an intracellular signal, although the cause of the signal can be extracellular: e.g., UV light exposure can induce DNA damage that leads to intrinsic apoptosis. The source of the damage comes from the outside, but the signal that starts the apoptotic process is the damage itself, which is intracellular. It is always mediated by mitochondrial events (and often referred to as “mitochondrial apoptosis pathway”). Children of this term refer to the particular type of signal that starts the apoptotic process. Annotation example: See PMID:21771788, annotations to ZPR9 (ZNF622, Q969S3) and ASK1 (MAP3K5, Q99683).

A diagram of the intrinsic apoptotic pathway including examples of molecular players can be found in Figure 2 in PMID:21760595.

Other children of apoptotic signaling pathway

There are events that escape the classification for extrinsic or intrinsic pathway of apoptosis, so they need specific terms and these have been placed under the more generic parent “apoptotic signaling pathway”:

[GO:0008626] granzyme-mediated apoptotic signaling pathway

Granzymes are proteolytic enzymes released from cytoplasmic granules produced by NK cells and T-cells. As such, they can activate effector caspases directly (prominently, caspase-3), by-passing the extrinsic and intrinsic pathways. They can also cleave proteins with a role in the intrinsic pathway, activating apoptosis through it.

[GO:0008628] hormone-mediated apoptotic signaling pathway

This term may be used to annotate apoptotic events triggered by hormones. Note that different hormones can activate apoptosis in different ways.

[GO:0008637] apoptotic mitochondrial changes and children

Due to the importance of the role of the mitochondrial compartment in apoptosis, this specific term was created to reflect events affecting it. When current knowledge allows for it, children and descendants of this term are linked to either the signaling or the execution phase of apoptosis, via “part_of” relationship. The following children of GO:0008637 deserve a specific comment:

[GO:0043653] mitochondrial fragmentation involved in apoptotic process

Although most of the processes described under ‘apoptotic mitochondrial changes’ take place during the signaling phase of apoptosis, this one cannot be confidently placed there. It is still controversial whether this process is involved in the signaling phase of apoptosis or not, so it was placed under the more generic apoptotic mitochondrial changes parent and it will not be linked to the signaling or the execution phase until further research clarifies the matter.

[GO:0097345] mitochondrial outer membrane permeabilization (MOMP) and children

This important mitochondrial event allows for the release of cytochrome c from the mitochondrial intermembrane space (see below) and the formation of the apoptosome, triggering the activation of caspase 9. It is therefore a key step in the apoptotic signaling pathway and it is sometimes monitored experimentally to detect apoptosis. Curators should be cautious when annotating to its regulatory terms, since pretty much any apoptosis-inducing event will trigger MOMP. Only events directly and specifically affecting this permeabilization should call for an annotation to MOMP or to its regulation terms. MOMP is initiated by the insertion of certain proteins into the mitochondrial membrane through a mechanism that is not yet fully understood. Gene products involved in this initiatory event can be annotated to the term [GO:0001844] “protein insertion into mitochondrial membrane involved in apoptotic signaling pathway”. Annotation example: See PMID:20850011, annotations to Drp1 (DNM1L, O00429).

[GO:0001836] release of cytochrome c from mitochondria

Even more than in the previous case, annotation to this term, and especially to its corresponding regulatory terms, should be done with caution. Monitoring the release of cytochrome c is a common way to detect when apoptosis is occurring and it is widely used in the literature. Only when experimental evidence is provided that a gene product directly and specifically affects this release, an annotation should be made to this term or to its regulation terms. Annotation example: See PMID:20850011, annotations to Drp1 (DNM1L, O00429).

[GO:0032976] release of matrix enzymes from mitochondria

Annotation to this term should be done with extreme caution, since it is not clear whether this is a recognized process in the scientific community. In the only annotation that the term has (PMID:9843949) the authors perform a couple of assays in which they detect the release of matrix enzymes, but they consider them as evidence of induction of the “permeability transition pore”, a controversial structure whose physiological significance is still unclear.

[GO:0097194] execution phase of apoptosis and children

Gene products involved in events that take place after initiator caspases have been activated and before the cell dies can be annotated to this term and to its following children:

[GO:0006921] cellular component disassembly involved in execution phase of apoptosis and children

This term and its children can be used to annotate for the breakdown of cellular structures during apoptosis.

[GO:0070782] phosphatidylserine exposure on apoptotic cell surface

This event acts as an “eat-me” signal for macrophages to dispose of the dying cell. Curators should be cautious when annotating to this term, since phosphatidylserine exposure on the cell surface can happen in other circumstances, not only in apoptosis. If no additional evidence of apoptotic process is given, the term should not be used.

Annotations to regulatory terms under [GO:0006915] “apoptotic process”

Apoptosis is a fundamental cellular response to different types of injury or aggression. As such, experimental assays knocking-down or over-expressing particular gene products will often trigger it, or diminish the cell’s ability to enter apoptosis. It is then tempting to annotate these gene products with the “positive/negative regulation of apoptotic process”-type terms. However, apoptosis can occur in these cases as a side effect of the disruption of a major pathway; if so, the gene products may not really be acting as direct, specific regulators of apoptosis, and the annotation would be incorrect. On the other hand, it is often difficult to ascertain if we are having a specific effect or not. Therefore, as a general rule of thumb, we would recommend avoiding annotating to regulatory terms for specific terms such as [GO:0090201] “negative regulation of release of cytochrome c from mitochondria”, unless evidence for the specificity of the regulation is given. In this case, more general terms, such as [GO:0006915] “apoptotic process”, allow for more flexibility in the annotation while still providing a link to the apoptotic process, and call for case-by-case decisions.

Annotations to caspase-related terms (“molecular function” branch of GO)

The word “caspase” was removed from all the term names related to this group of enzymes because it represents a gene product and because the distinction from other types of proteases is outside the scope of GO. Since the activity of caspases can be described as a “cysteine-type endopeptidase activity”, this wording replaces “caspase” wherever it was used in GO. See e.g. discussion in the following link: https://sourceforge.net/tracker/?func=detail&aid=3307514&group_id=36855&atid=440764. There are terms such as [GO:0006919] “activation of cysteine-type endopeptidase activity involved in apoptotic process” that specifically refer to a particular instance of the positive regulation of caspase activity. The aim of these terms is to capture the complexity of the caspase activity regulation process. Since caspases are synthesized as zymogens and then cleaved into active proteases, there is a need for terms that can capture both the activation process and the regulation of the enzymatic activity part. This way, proteins that directly perform or enable the cleavage of pro-caspases into their active, mature forms may be annotated using this term. If they increase the activity of an already active caspase or they activate it in an indirect fashion, they should be annotated using the parent term [GO:0043280] “positive regulation of cysteine-type endopeptidase activity involved in apoptotic process”. Only the positive regulation terms are mentioned here as guidance, but there are negative (inhibiting) ones as well when it applies.