Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

The ATM kinase and p53 are key tumor suppressor factors that control the genotoxic stress response pathway. The ATM substrate Mdm2 controls p53 activity by either targeting p53 for degradation or promoting its synthesis by binding the p53 mRNA. The physiological role and regulation of Mdm2's dual function toward p53 is not known. Here we show that ATM-dependent phosphorylation of Mdm2 at Ser395 is required for the p53 mRNA-Mdm2 interaction. This event also promotes SUMO-conjugation of Mdm2 and its nucleoli accumulation. Interfering with the p53 mRNA-Mdm2 interaction prevents p53 stabilization and activation following DNA damage. These results demonstrate how ATM activity switches Mdm2 from a negative to a positive regulator of p53 via the p53 mRNA.

MDM2 is considered a hub protein due to its capacity to interact with a large number of different partners of which p53 is most well described. MDM2 is an E3 ubiquitin ligase, and many, but not all, of its interactions relate directly to this activity, such as substrates, adaptors or bridges, promoters, inhibitors or complementary factors. Some interactions serve regulatory functions that in response to cellular stresses control the localisation and functions of MDM2 including protein kinases, ribosomal proteins and proteases. Moreover, interactions with nucleotides serve other functions such as mRNA to regulate protein synthesis and DNA to control transcription. To perform such a pleiotropic panorama of different functions, MDM2 is subjected to a multitude of post-translational modifications and is expressed in different isoforms. The large and diverse interactome is made possible due to the plasticity of MDM2 and in this review we have listed the MDM2 interactions until now and we will discuss how this multifaceted protein can interact with such a variety of substrates to provide a key intermediary role in different signalling pathways.

A large number of signalling pathways converge on p53 to induce different cellular stress responses that aim to promote cell cycle arrest and repair or, if the damage is too severe, to induce irreversible senescence or apoptosis. The differentiation of p53 activity towards specific cellular outcomes is tightly regulated via a hierarchical order of post-translational modifications and regulated protein-protein interactions. The mechanisms governing these processes provide a model for how cells optimize the genetic information for maximal diversity. The p53 mRNA also plays a role in this process and this review aims to illustrate how protein and RNA interactions throughout the p53 mRNA in response to different signalling pathways control RNA stability, translation efficiency or alternative initiation of translation. We also describe how a p53 mRNA platform shows riboswitch-like features and controls the rate of p53 synthesis, protein stability and modifications of the nascent p53 protein. A single cancer-derived synonymous mutation disrupts the folding of this platform and prevents p53 activation following DNA damage. The role of the p53 mRNA as a target for signalling pathways illustrates how mRNA sequences have co-evolved with the function of the encoded protein and sheds new light on the information hidden within mRNAs.

We examined the interfaces of homodimeric triosephosphate isomerase (TIM) from eight different species. The crystal structures of the enzymes showed that a portion of the interface is markedly similar in TIMs from Trypanosoma cruzi (TcTIM), Trypanosoma brucei, and Leishmania mexicana and significantly different from that of TIMs from human, yeast, chicken, Plasmodium falciparum, and Entamoeba histolytica. Since this interfacial region is central in the stability of TcTIM, we hypothesized that it would be possible to find agents that selectively affect the stability of TIMs from the three trypanosomatids. We found that 6,6'-bisbenzothiazole-2,2' diamine in the low micromolar range causes a desirable irreversible inactivation of the enzymes from the three trypanosomatids and has no effect on the other five TIMs. Thus, the data indicate that it is possible to find compounds that induce selective inactivation of the enzymes from three different trypanosomatids.

No abstract

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.