Cyclin C: The Story of a Non-Cycling Cyclin.

The class I cyclin family is a well-studied group of structurally conserved proteins that interact with their associated cyclin-dependent kinases (Cdks) to regulate different stages of cell cycle progression depending on their oscillating expression levels. However, the role of class II cyclins, which primarily act as transcription factors and whose expression remains constant throughout the cell cycle, is less well understood. As a classic example of a transcriptional cyclin, cyclin C forms a regulatory sub-complex with its partner kinase Cdk8 and two accessory subunits Med12 and Med13 called the Cdk8-dependent kinase module (CKM). The CKM reversibly associates with the multi-subunit transcriptional coactivator complex, the Mediator, to modulate RNA polymerase II-dependent transcription. Apart from its transcriptional regulatory function, recent research has revealed a novel signaling role for cyclin C at the mitochondria. Upon oxidative stress, cyclin C leaves the nucleus and directly activates the guanosine 5'-triphosphatase (GTPase) Drp1, or Dnm1 in yeast, to induce mitochondrial fragmentation. Importantly, cyclin C-induced mitochondrial fission was found to increase sensitivity of both mammalian and yeast cells to apoptosis. Here, we review and discuss the biology of cyclin C, focusing mainly on its transcriptional and non-transcriptional roles in tumor promotion or suppression.

Cyclin C directly stimulates Drp1 GTP affinity to mediate stress-induced mitochondrial hyperfission.

Mitochondria exist in an equilibrium between fragmented and fused states that shifts heavily toward fission in response to cellular damage. Nuclear-to-cytoplasmic cyclin C relocalization is essential for dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in response to oxidative stress. This study finds that cyclin C directly interacts with the Drp1 GTPase domain, increases its affinity to GTP, and stimulates GTPase activity in vitro. In addition, the cyclin C domain that binds Drp1 is contained within the non-Cdk binding second cyclin box domain common to all cyclin family members. This interaction is important, as this domain is sufficient to induce mitochondrial fission when expressed in mouse embryonic fibroblasts in the absence of additional stress signals. Using gel filtration chromatography and negative stain electron microscopy, we found that cyclin C interaction changes the geometry of Drp1 oligomers in vitro. High-molecular weight low-GTPase activity oligomers in the form of short filaments and rings were diminished, while dimers and elongated filaments were observed. Our results support a model in which cyclin C binding stimulates the reduction of low-GTPase activity Drp1 oligomers into dimers capable of producing high-GTPase activity filaments.

A complex molecular switch directs stress-induced cyclin C nuclear release through SCFGrr1-mediated degradation of Med13.

In response to oxidative stress, cells decide whether to mount a survival or cell death response. The conserved cyclin C and its kinase partner Cdk8 play a key role in this decision. Both are members of the Cdk8 kinase module, which, with Med12 and Med13, associate with the core mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which thereafter releases cyclin C into the cytoplasm. Cytoplasmic cyclin C associates with mitochondria, where it induces hyperfragmentation and regulated cell death. In this report, we show that residues 742-844 of Med13's 600-amino acid intrinsic disordered region (IDR) both directs cyclin C-Cdk8 association and serves as the degron that mediates ubiquitin ligase SCFGrr1-dependent destruction of Med13 following oxidative stress. Here, cyclin C-Cdk8 phosphorylation of Med13 most likely primes the phosphodegron for destruction. Next, pro-oxidant stimulation of the cell wall integrity pathway MAP kinase Slt2 initially phosphorylates cyclin C to trigger its release from Med13. Thereafter, Med13 itself is modified by Slt2 to stimulate SCFGrr1-mediated destruction. Taken together, these results support a model in which this IDR of Med13 plays a key role in controlling a molecular switch that dictates cell fate following exposure to adverse environments.

Ceramide channel: Structural basis for selective membrane targeting.

A ceramide commonly found in mammalian cells, C16-ceramide (N-palmitoyl-d-erythro-sphingosine), is capable of forming large, protein-permeable channels in the mitochondrial outer membrane (MOM). However, C16-ceramide is unable to permeabilize the plasma membrane of erythrocytes. This specificity is unexpected considering that ceramide forms channels in simple phosphoglycerolipid membranes. Synthetic analogs of C16-ceramide with targeted changes at each of the functional regions of the molecule including methylation, altered hydrocarbon chain length, and changes in the stereochemistry, were tested to probe the role of ceramide's molecular features on its ability to form channels in these two different membrane types. The ability to permeabilize the MOM was relatively insensitive to modifications of the various functional groups of ceramide whereas the same modifications resulted in plasma membrane permeabilization (a gain of function rather than a loss of function). Some analogs (ceramine, NBD-labeled ceramide, C18,1 ceramide) gained another function, the ability to inhibit cytochrome oxidase. The gain of deleterious functions indicates that constraints on the structure of ceramide that is formed by the cell's synthetic machinery includes the avoidance of deleterious interactions. We propose that the specific structure of ceramide limits the size of its interactome (both proteins and lipids) thus reducing the likelihood of unwanted side effects.

The dynamics of Bax channel formation: influence of ionic strength.

Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, detected by protein release, and more substantial long-lasting permeabilization, measured by the rate of oxidation of added cytochrome c. We found that higher ionic strength causes Bax to form small channels quickly but the expansion of these early channels is impeded. This inhibitory effect of ionic strength is independent of tBid. Channels formed under low ionic strength are not destabilized by raising the ionic strength. Increase in ionic strength also increases the ability of Bcl-xL to inhibit Bax-mediated MOMP. Ionic strength does not affect Bax insertion into mitochondria. Thus, ionic strength influences the assembly of Bax molecules already in membrane into channels. Ionic strength can be used as an effective biophysical tool to study Bax-mediated channel formation.

Ceramide channels: influence of molecular structure on channel formation in membranes.

The sphingolipid, ceramide, self-assembles in the mitochondrial outer membrane (MOM), forming large channels capable of translocating proteins. These channels are believed to be involved in protein release from mitochondria, a key decision-making step in cell death. Synthetic analogs of ceramide, bearing modifications in each of the major structural features of ceramide were used to probe the molecular basis for the stability of ceramide channels. Channel stability and mitochondrial permeabilization were disrupted by methylation of the C1-hydroxyl group whereas modifications of the C3 allylic hydroxyl group were well tolerated. A change in chirality at C2 that would influence the orientation of the C1-hydroxyl group resulted in a strong reduction of channel-forming ability. Similarly, methylation of the amide nitrogen is also detrimental to channel formation. Many changes in the degree, location and nature of the unsaturation of ceramide had little effect on mitochondrial permeabilization. Competition experiments between ceramide and analogs resulted in synergy with structures compatible with the ceramide channel model and antagonism with incompatible structures. The results are consistent with ceramide channels being highly organized structures, stabilized by specific inter-molecular interactions, similar to the interactions responsible for protein folding.

Regulation of ceramide channels by Bcl-2 family proteins.

Mitochondrial outer membrane permeabilization to proteins, an irreversible step in apoptosis by which critical proteins are released, is tightly regulated by Bcl-2 family proteins. The exact nature of the release pathway is still undefined. Ceramide is an important sphingolipid, involved in various cellular processes including apoptosis. Here we describe the structural properties of ceramide channels and their regulation by the anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. The evolutionarily conserved regulation of ceramide channels by Bcl-2 family proteins, consistent with their role in apoptosis, lends credibility to the notion that ceramide channels constitute the protein release pathway.

Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane.

A critical step in apoptosis is mitochondrial outer membrane permeabilization (MOMP), releasing proteins critical to downstream events. While the regulation of this process by Bcl-2 family proteins is known, the role of ceramide, which is known to be involved at the mitochondrial level, is not well-understood. Here, we demonstrate that Bax and ceramide induce MOMP synergistically. Experiments were performed on mitochondria isolated from both rat liver and yeast (lack mammalian apoptotic machinery) using both a protein release assay and real-time measurements of MOMP. The interaction between activated Bax and ceramide was also studied in a defined isolated system: planar phospholipid membranes. At concentrations where ceramide and activated Bax have little effects on their own, the combination induces substantial MOMP. Direct interaction between ceramide and activated Bax was demonstrated both by using yeast mitochondria and phospholipid membranes. The apparent affinity of activated Bax for ceramide increases with ceramide content indicating that activated Bax shows enhanced propensity to permeabilize in the presence of ceramide. An agent that inhibits ceramide-induced but not activated Bax induced permeabilization blocked the enhanced MOMP, suggesting that ceramide is the key permeabilizing entity, at least when ceramide is present. These and previous findings that anti-apoptotic proteins disassemble ceramide channels suggest that ceramide channels, regulated by Bcl-2-family proteins, may be responsible for the MOMP during apoptosis.