Apc C Triggers Anaphase By Marking The Protein For Degradation

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that orchestrates the metaphase-anaphase transition during cell division by targeting specific proteins for degradation via the ubiquitin-proteasome system. This process is crucial for ensuring accurate chromosome segregation, preventing aneuploidy, and maintaining genomic stability. The APC/C triggers anaphase by ubiquitinating key regulatory proteins, marking them for degradation by the 26S proteasome. This article delves into the intricate mechanisms by which the APC/C functions, the specific proteins it targets, and the consequences of its misregulation.

Introduction to the Anaphase-Promoting Complex/Cyclosome (APC/C)

The APC/C is a large multi-subunit complex that acts as a master regulator of the cell cycle, particularly the transition from metaphase to anaphase. It is responsible for the timely and irreversible separation of sister chromatids, which is a critical step in cell division. The APC/C functions as an E3 ubiquitin ligase, meaning it facilitates the transfer of ubiquitin molecules to target proteins, thereby tagging them for degradation by the 26S proteasome.

The activation of the APC/C is tightly controlled and occurs in a sequential manner, involving several regulatory proteins and checkpoints. This ensures that anaphase is initiated only when all chromosomes are correctly attached to the spindle microtubules, preventing errors in chromosome segregation. The APC/C’s activity is essential not only for the metaphase-anaphase transition but also for regulating other cell cycle events, such as mitotic exit and the initiation of DNA replication in the subsequent cell cycle.

Structure and Activation of the APC/C

The APC/C is a complex consisting of at least 19 different subunits in yeast and mammals, forming a highly intricate molecular machine. These subunits can be broadly categorized into structural components, catalytic components, and regulatory components.

Structural Components: Provide a scaffold for the complex and facilitate its assembly.
Catalytic Components: Include the core ubiquitin ligase components, such as APC2 and APC11, which are responsible for the transfer of ubiquitin to target proteins.
Regulatory Components: Mediate the activation and substrate specificity of the APC/C.

The APC/C is activated by associating with one of two activating subunits: Cdc20 (Cell division cycle protein 20) in early mitosis and Cdh1 (Cdc20 homolog 1) in late mitosis and G1 phase. These activating subunits are essential for substrate recognition and binding.

Cdc20: Primarily active during metaphase, promotes the degradation of securin and cyclin B.
Cdh1: Becomes active in late mitosis and G1 phase, maintaining the APC/C activity and ensuring that cells remain in a quiescent state until the next cell cycle.

The activation of APC/C-Cdc20 is tightly regulated by the spindle assembly checkpoint (SAC), which monitors the attachment of chromosomes to the mitotic spindle. The SAC prevents premature anaphase onset by inhibiting APC/C-Cdc20 until all chromosomes are properly attached. Once the SAC is satisfied, APC/C-Cdc20 is activated, leading to the ubiquitination and degradation of its target proteins.

The Ubiquitin-Proteasome System and Protein Degradation

The ubiquitin-proteasome system (UPS) is the major pathway for selective protein degradation in eukaryotic cells. It plays a crucial role in regulating a wide range of cellular processes, including cell cycle progression, signal transduction, and DNA repair. The UPS involves two main steps:

Ubiquitination: The process of attaching ubiquitin molecules to target proteins.
Proteasomal Degradation: The degradation of ubiquitinated proteins by the 26S proteasome.

Ubiquitination is a multi-step enzymatic process that involves three main enzymes:

E1 (Ubiquitin-Activating Enzyme): Activates ubiquitin in an ATP-dependent manner.
E2 (Ubiquitin-Conjugating Enzyme): Transfers ubiquitin from E1 to E3.
E3 (Ubiquitin Ligase): Recognizes the target protein and facilitates the transfer of ubiquitin from E2 to the target protein.

The APC/C is an E3 ubiquitin ligase that specifically targets proteins involved in cell cycle regulation. It catalyzes the formation of a polyubiquitin chain on its target proteins, which serves as a signal for recognition and degradation by the 26S proteasome.

The 26S proteasome is a large multi-subunit complex that degrades ubiquitinated proteins into small peptides. It consists of a 20S core particle, which contains the proteolytic active sites, and two 19S regulatory particles, which recognize and unfold ubiquitinated proteins.

Key Target Proteins of APC/C

The APC/C targets several key regulatory proteins for degradation, which are essential for the metaphase-anaphase transition and mitotic exit. The two primary targets of APC/C are securin and cyclin B.

Securin

Securin is an inhibitory protein that binds to and inhibits separase, a protease responsible for cleaving cohesin. Cohesin is a protein complex that holds sister chromatids together until anaphase. By inhibiting separase, securin prevents premature separation of sister chromatids.

The APC/C, when activated by Cdc20 (APC/C-Cdc20), ubiquitinates securin, marking it for degradation by the 26S proteasome. The degradation of securin releases separase, which then cleaves cohesin, allowing sister chromatids to separate and move to opposite poles of the cell.

The degradation of securin is a critical step in initiating anaphase and ensuring accurate chromosome segregation. Failure to degrade securin can lead to premature sister chromatid separation, resulting in aneuploidy and genomic instability.

Cyclin B

Cyclin B is a regulatory subunit of the M-phase promoting factor (MPF), also known as cyclin-dependent kinase 1 (Cdk1). MPF is a key regulator of entry into and progression through mitosis. Cyclin B binds to Cdk1 and activates its kinase activity, which phosphorylates numerous target proteins, driving the cell into mitosis.

The APC/C, both when activated by Cdc20 (APC/C-Cdc20) and Cdh1 (APC/C-Cdh1), ubiquitinates cyclin B, marking it for degradation by the 26S proteasome. The degradation of cyclin B leads to inactivation of Cdk1, which is essential for mitotic exit and the transition to the next cell cycle.

The degradation of cyclin B is crucial for several reasons:

Mitotic Exit: Inactivation of Cdk1 is necessary for the cell to exit mitosis and enter the G1 phase.
Cytokinesis: Cdk1 inactivation promotes cytokinesis, the physical division of the cell into two daughter cells.
Prevention of Re-entry into Mitosis: Maintaining low levels of Cdk1 activity in G1 prevents premature entry into mitosis.

Other Targets

Besides securin and cyclin B, the APC/C also targets other proteins involved in cell cycle regulation. These include:

Aurora B Kinase: Involved in chromosome segregation and cytokinesis. APC/C-Cdh1 promotes the degradation of Aurora B kinase during mitotic exit.
Polo-like Kinase 1 (Plk1): Regulates various aspects of mitosis, including spindle formation and chromosome segregation. APC/C-Cdh1 mediates the degradation of Plk1 during mitotic exit.
Nek2: A kinase involved in centrosome separation and spindle formation. APC/C-Cdh1 targets Nek2 for degradation during mitotic exit.

Molecular Mechanisms of APC/C-Mediated Ubiquitination

The APC/C recognizes its target proteins through specific degrons, short amino acid sequences that serve as binding sites for the APC/C activators, Cdc20 and Cdh1. The two main degrons recognized by APC/C are the Destruction box (D-box) and the KEN box.

Destruction Box (D-box): A short sequence of amino acids, typically RXXLXXXXN (where X is any amino acid), found in many APC/C substrates, including securin and cyclin B.
KEN Box: A three-amino acid sequence (KEN) found in some APC/C substrates, such as Cdh1 and Plk1.

The D-box and KEN box are recognized by Cdc20 and Cdh1, respectively, which then recruit the APC/C to the target protein. Once the target protein is bound to the APC/C, the ubiquitin ligase activity of the APC/C is activated, leading to the addition of ubiquitin molecules to the target protein.

The ubiquitination process typically involves the formation of a polyubiquitin chain, where multiple ubiquitin molecules are linked together. The most common type of polyubiquitin chain is linked through lysine 48 (K48) of ubiquitin, which serves as a signal for degradation by the 26S proteasome.

Regulation of APC/C Activity

The activity of the APC/C is tightly regulated by various mechanisms, including:

Spindle Assembly Checkpoint (SAC): As mentioned earlier, the SAC inhibits APC/C-Cdc20 until all chromosomes are properly attached to the mitotic spindle. The SAC is activated by unattached or misattached chromosomes, which generate a signal that inhibits APC/C-Cdc20.
Phosphorylation: The APC/C and its activators, Cdc20 and Cdh1, are regulated by phosphorylation. For example, Cdk1 phosphorylation of APC/C subunits can modulate its activity.
APC/C Inhibitory Proteins: Several proteins can directly inhibit the APC/C, such as Emi1 (Early mitotic inhibitor 1), which binds to and inhibits APC/C-Cdc20 during early mitosis.
Subcellular Localization: The localization of the APC/C and its activators can also regulate its activity. For example, Cdh1 is sequestered in the cytoplasm during early mitosis, preventing its interaction with the APC/C.

Consequences of APC/C Misregulation

Misregulation of the APC/C can have severe consequences for cell division and genomic stability. Failure to properly regulate the APC/C can lead to:

Aneuploidy: Incorrect chromosome segregation, resulting in cells with an abnormal number of chromosomes. Aneuploidy is a hallmark of cancer cells and can contribute to tumorigenesis.
Premature Sister Chromatid Separation: Degradation of cohesin before proper chromosome alignment, leading to chromosome missegregation.
Mitotic Exit Defects: Failure to degrade cyclin B, resulting in prolonged mitosis and potential cell death.
Genomic Instability: Accumulation of DNA damage and mutations due to errors in cell division.

Several human diseases are associated with APC/C misregulation, including:

Cancer: Mutations in APC/C subunits or regulatory proteins have been identified in various types of cancer. For example, mutations in APC (Adenomatous polyposis coli), a tumor suppressor protein that regulates APC/C activity, are common in colorectal cancer.
Developmental Disorders: APC/C misregulation can disrupt normal development, leading to birth defects and developmental abnormalities.
Neurodegenerative Diseases: Accumulating evidence suggests that APC/C dysfunction may contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Therapeutic Implications

Given the critical role of the APC/C in cell division and its involvement in various diseases, the APC/C has emerged as a potential therapeutic target. Several strategies are being explored to modulate APC/C activity for therapeutic purposes:

Small Molecule Inhibitors: Development of small molecules that directly inhibit the APC/C. These inhibitors could be used to selectively kill cancer cells by disrupting their cell cycle progression.
Targeting APC/C Regulators: Targeting proteins that regulate APC/C activity, such as Cdc20 and Cdh1. Inhibiting these regulators could indirectly modulate APC/C activity and disrupt cell cycle progression.
Restoring APC/C Function: In cases where APC/C function is impaired due to mutations, therapeutic strategies could focus on restoring APC/C activity. This could involve gene therapy or the development of drugs that enhance APC/C function.

Future Directions and Research

The APC/C is a complex and fascinating molecular machine that plays a central role in cell division. Despite significant progress in understanding the APC/C, many questions remain unanswered. Future research directions include:

Detailed Structural Analysis: Obtaining high-resolution structures of the APC/C in different functional states to understand its mechanism of action at the atomic level.
Identification of Novel APC/C Targets: Identifying new proteins that are targeted by the APC/C to gain a more comprehensive understanding of its role in cell cycle regulation.
Regulation of APC/C in Different Cell Types: Investigating how the APC/C is regulated in different cell types and developmental stages to understand its diverse functions.
Role of APC/C in Disease Pathogenesis: Elucidating the role of APC/C misregulation in various diseases and developing therapeutic strategies to target the APC/C.

Conclusion

The anaphase-promoting complex/cyclosome (APC/C) is a master regulator of the cell cycle that triggers anaphase by ubiquitinating key regulatory proteins and marking them for degradation. This process is essential for accurate chromosome segregation, preventing aneuploidy, and maintaining genomic stability. The APC/C targets securin and cyclin B, among other proteins, for degradation via the ubiquitin-proteasome system. The activity of the APC/C is tightly regulated by the spindle assembly checkpoint, phosphorylation, and inhibitory proteins. Misregulation of the APC/C can lead to aneuploidy, mitotic exit defects, and genomic instability, contributing to diseases such as cancer and developmental disorders. The APC/C has emerged as a potential therapeutic target, and future research will continue to unravel its intricate mechanisms and role in disease pathogenesis. The understanding of APC/C’s function provides critical insights into cell cycle control and opens new avenues for therapeutic interventions.