Assembling Multiprotein Complexes

Histone acetyltransferases (HATs) are enzymes that acetylate specific lysine residues in histones through the transfer of an acetyl group from an acetyl-coenzymeA (AcCoA) molecule, causing profound effects on chromatin structure and assembly as well as gene transcription. HATs are found in most, if not all, eukaryotic organisms as multiprotein complexes, some HAT catalytic subunits even being shared between various complexes that display different substrate specificities based on their subunit composition [12]. Despite their name, HATs do not restrict themselves to the acetylation of histones, since these enzymes have also been shown to act on nonhistone proteins, broadening their scope of action [13]. 

Hydrophobic and osmophobic effects are important not only in the folding of individual polypeptide chains into compact globular proteins, but also in the assembly of multiprotein complexes. Osmophobic effects are noted, for instance, in the self-assembly of subunits of the glycolytic enzyme phosphofructokinase (PFK). Self-assembly is enhanced by the presence of stabilizing organic cosolvents such as trimethylamine-A-oxide (TMAO) (Hand and Somero, 1982). As discussed later, self-assembly driven by osmophobic effects results from the thermodynamic favorability of minimizing the surface area on the proteins that is in contact with the cosolvent. 

Several families of transcription factors exist. These include basal (or general) transcription factors, activators, and repressors. The basal transcription factors include transcription factor IIA (TFUA), TFIIB, TRID, TFIIE, TRIP, and TFllH. Most of these transcription factors exist as multiprotein complexes. These transcription factors must be assembled just upstream of the transcribed part of the gene before RNA polymerase begins its catalytic activity. When assembled, the entire "mega-complex" consists of about 50 proteins (Jacobson and Tjian, 1996). 

The picture of the structure and function of TFIIH is thus varied and complex. TFIIH or components of it can assemble into different multiprotein complexes which perfom central functions in the cell. The mechanistic details and the regulation of the various activities have long been not understood. 

The colocalization of signaling proteins is essential for the signaling at the cell membrane. The transduction of signals by transmembrane receptors into the interior of the cell occurrs in multiprotein complexes assembled specifically at the inner face of the cell membrane. A large part of the Ras signaling pathway (see Chapter 9) is intimately linked to the cytoplasmic side of the membrane. If membrane association of the components of the Ras signaling pathway is prevented, signal transduction is shut down. 

The initiator caspases receive proapoptotic signals and initiate the activation of a caspase cascade. They are activated by assembly into a multiprotein complex, and they contain large prodomains responsible for this interaction. 

In this pathway, an apoptotic stimulus induces assembly of the procaspase into a multiprotein complex. Activation is assumed to occur by autoproteolyis involving mutual cleavage in trans within this complex. Specific cofactors like FADD (see Section 15.6) and Apafl/Cytc (see Section 15.5) are involved in the formation of... 

Recruitment of the initiator procaspases into a multiprotein complex results from a regulated series of protein-protein interactions mediated by interaction modules . Four types of interaction modules are involved in the activation of initiator caspases and thus play important roles in the initiation of apoptosis (review Weber and Vin-cenz, 2001). These domains have been named the death domain (DD),, the death effector domain (DED), the caspase activation and recruitment domain (CARD), and the less characterized pyrin domain. The domains are found on several components of the apoptotic signaling pathways and mediate homotypic protein-protein interactions, i. e., a given module will interact only with a member of the same family and not with members of the other families. Since members of the same module are found on different proteins, these modules mediate the assembly of hetero-oligomeric protein complexes. As examples, DDs are found on death receptors and their cofactors, D EDs on cofactors and the initiator caspase-8, and CARDS on cofactors, caspase-2, and caspase-9. 

In a further step of apoptosis, the cytochrome c released from the mitochondria promotes the assembly of a multiprotein complex, termed apoptosome, which contains cytochrome c, the adaptor protein Apafl, and procaspase-9. The apoptosome requires ATP for its formation and is able to cleave and activate procaspase-3, an effector caspase. The adaptor protein Apafl appears to play a major structural role in this assembly. Apafl contains WD motifs for interaction with cytochrome c and a CARD motif, which directs binding to the CARD motifs of procaspase-9 and procaspase-3. Structural studies on the apoptosome by electron microscopy have revealed a wheel shaped heptameric complex, with the CARD domains of Apafl located at the central hub and the WD40 repeats at the extended spokes (Acehan et al., 2002). The location of pro-caspase 9 in this complex is still open as is the mechanism of caspase 9 activation. 

Fig. 15.11 Si gnaling by the tumor necrosis factor (TNF) receptor. Binding of TNF to its receptor induces association and activation for further signaling of several proteins which activate distinct signaling pathways. Assembly of the multiprotein complex on the cytoplasmic side is mediated mainly via death domains (DD) ofthe receptor and the adaptor protein TRADD. FADD induces apoptosis via activation of initiator caspase 8. TRAF2 and RIP mediate activation of transcription via two main ways. One way uses phosphorylation ofthe inhibitor IkB by IkB kinase (IKK) to induce its ubiquitin-mediated proteolytic destruction and the relieve ofNF cB inhibition. Another way leads to activation ofthe JNK pathway (see Chapter 10) and stimulation of transcription of diverse target genes.

Cooperative binding of multiple activators to nearby sites in an enhancer forms a multiprotein complex called an enhancesome (see Figure 11-26). Assembly of en-hancesomes often requires small proteins that bind to the DNA minor groove and bend the DNA sharply, allowing bound proteins on either side of the bend to interact more readily. 

Assembly of the large, multiprotein cleavage/polyadenyl-ation complex around the AU-rIch poly(A) signal In a pre-mRNA is analogous In many ways to formation of the transcriptlon-prelnitlatlon complex at the AT-rIch TATA box of a template DNA molecule (see Figure 11-27). In both cases, multiprotein complexes assemble cooperatively through a network of specific protein-nucleic acid and protein-protein Interactions. 

Receptors Frizzled (Fz) with seven transmembrane a helices associated membrane-bound LDL receptor-related protein (Lrp) required for receptor activity Signal transduction Assembly of multiprotein complex at membrane that inhibits the proteasome-mediated proteolysis of cytosolic p-catenin transcription factor, resulting in its accumulation... 

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