PUMA cooperates with direct activator proteins to promote mitochondrial outer membrane permeabilization and apoptosis

The BCL-2 family of proteins regulates apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP). Within the family there are numerous protein?protein interactions that influence MOMP; however, defining the ultimate signal that commits a cell to apoptosis remains controversial. We chose to examine the function of the BH3-only protein, p53 upregulated modulator of apoptosis (PUMA), to define its contribution to MOMP and cooperation with the direct activator proteins. PUMA is a potent regulator of MOMP and our data suggest that this function is attributed to two distinct mechanisms which both rely on PUMA binding to the anti-apoptotic BCL-2 proteins: de-repression and sensitization. Here we will define these interactions and discuss our experiments that suggest PUMA cooperates with direct activator proteins to efficiently induce MOMP and apoptosis.     

Novel protein interactors of urokinase-type plasminogen activator receptor

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.     

Protein-protein interactions in Cdk5 regulation and function

Cdk5 is a unique member of the cyclin-dependent kinase (Cdk) family of small protein kinases. In association with its neuron-specific activator p35 or p39, Cdk5 displays many regulatory properties distinct from other Cdks. A growing body of evidence has suggested that Cdk5-p35 has important implications in a variety of neuronal activities occurring in the central nervous system. In brain, Cdk5-p35 appears to exist as large molecular complexes with other proteins, and protein-protein interactions appear to be a molecular principle for Cdk5-p35 to conduct its physiological functions. Over the past decade, a number of proteins have been identified to associate with Cdk5-p35. While the majority of these proteins mediate their interaction with Cdk5 through p35, implying that p35 may act not only as an activator of Cdk5 but also as an adaptor to associate Cdk5 with its regulators and physiological targets, a small group of other proteins are found to link directly with Cdk5. In addition, Cdk5 has been found to phosphorylate a diverse list of substrates, further implicating its regulatory roles in a wide range of cellular processes. In this review, we present an updated inventory of the interacting proteins of Cdk5-p35 kinase and its substrates as well as a discussion on the implicated effects of these interactions.     

Calcium-dependent regulation of NAD kinase.

An activator protein of NAD kinase from the pea, $\text{Pisum}\text{satavum}$ L., has been shown to be Ca²⁺-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca²⁺-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, $\text{Renilla}$, will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.     

Protein kinase C binding partners

Members of the protein kinase C family respond to second messengers and are involved in controlling a broad array of cellular functions. The overlapping specificity and promiscuity of these proteins has promoted the view that specific binding proteins constrain individual family members to create the appropriate specificity of action. It is speculated that such protein kinase C-regulator protein interactions affect substrate availability as well as exposure to allosteric activator(s) and that consequent interactions specify cellular location and impose integration with other signaling systems. These predicted features have been realized in the identification of many protein kinase C interacting proteins and examples of these are discussed.     

On the impact of the distance between two genes on their interaction curve

We analyze a basic building block of gene regulatory networks using a stochastic/geometric model in search of a mathematical backing for the discrete modeling frameworks. We consider a network consisting only of two interacting genes: a source gene and a target gene. The target gene is activated by the proteins encoded by the source gene. The interaction is therefore mediated by activator proteins that travel, like a signal, from the source to the target. We calculate the production curve of the target proteins in response to a constant-rate production of activator proteins. The latter has a sigmoidal shape (like a simple delay line) that is sharper and taller when the two genes are closer to each other. This provides further support for the use of discrete models in the analysis gene regulatory networks. Moreover, it suggests an evolutionary pressure towards making the interacting genes closer to each other to make their interactions more efficient and more reliable.     

Mutation to Bax beyond the BH3 domain disrupts interactions with pro-survival proteins and promotes apoptosis

Pro-survival members of the Bcl-2 family of proteins restrain the pro-apoptotic activity of Bax, either directly through interactions with Bax or indirectly by sequestration of activator BH3-only proteins, or both. Mutations in Bax that promote apoptosis can provide insight into how Bax is regulated. Here, we describe crystal structures of the pro-survival proteins Mcl-1 and Bcl-x(L) in complex with a 34-mer peptide from Bax that encompasses its BH3 domain. These structures reveal canonical interactions between four signature hydrophobic amino acids from the BaxBH3 domain and the BH3-binding groove of the pro-survival proteins. In both structures, Met-74 from the Bax peptide engages with the BH3-binding groove in a fifth hydrophobic interaction. Various Bax Met-74 mutants disrupt interactions between Bax and all pro-survival proteins, but these Bax mutants retain pro-apoptotic activity. Bax/Bak-deficient mouse embryonic fibroblast cells reconstituted with several Bax Met-74 mutants are more sensitive to the BH3 mimetic compound ABT-737 as compared with cells expressing wild-type Bax. Furthermore, the cells expressing Bax Met-74 mutants are less viable in colony assays even in the absence of an external apoptotic stimulus. These results support a model in which direct restraint of Bax by pro-survival Bcl-2 proteins is a barrier to apoptosis.     

Juxtaposition between activation and basic domains of human immunodeficiency virus type 1 Tat is required for optimal interactions between Tat and TAR.

trans activation of the human immunodeficiency virus type 1 long terminal repeat requires that the viral trans activator Tat interact with the trans-acting responsive region (TAR) RNA. Although the N-terminal 47 amino acids represent an independent activation domain that functions via heterologous nucleic acid-binding proteins, sequences of Tat that are required for interactions between Tat and TAR in cells have not been defined. Although in vitro binding studies suggested that the nine basic amino acids from positions 48 to 57 in Tat bind efficiently to the 5' bulge in the TAR RNA stem-loop, by creating several mutants of Tat and new hybrid proteins between Tat and the coat protein of bacteriophage R17, we determined that this arginine-rich domain is not sufficient for interactions between Tat and TAR in vivo. Rather, the activation domain is also required and must be juxtaposed to the basic domain. Thus, in vitro TAR RNA binding does not translate to function in vivo, which suggests that other proteins are important for specific and productive interactions between Tat and TAR.