Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane.

Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair.

A phage protein-derived antipathogenic peptide that targets type IV pilus assembly.

Phage-inspired antibacterial discovery is a new approach that recruits phages in search for antibacterials with new molecular targets, in that phages are the biological entities well adapted to hijack host bacterial physiology in favor of their own thrive. We previously observed that phage-mediated twitching motility inhibition was effective to control the acute infections caused by Pseudomonas aeruginosa and that the motility inhibition was attributed to the delocalization of PilB, the type IV pilus (TFP) assembly ATPase by binding of the 136-amino acid (aa) phage protein, Tip. Here, we created a series of truncated and point-mutant Tip proteins to identify the critical residues in the Tip bioactivity: N-terminal 80-aa residues were dispensable for the Tip activity; we identified that Asp82, Leu84, and Arg85 are crucial in the Tip function. Furthermore, a synthetic 15-aa peptide (P1) that corresponds to Leu73 to Ala87 is shown to suffice for PilB delocalization, twitching inhibition, and virulence attenuation upon exogenous administration. The transgenic flies expressing the 15-aa peptide were resistant to P. aeruginosa infections as well. Taken together, this proof-of-concept study reveals a new antipathogenic peptide hit targeting bacterial motility and provides an insight into antibacterial discovery targeting TFP assembly.

Molecular mechanism of NFAT family proteins for differential regulation of the IL-2 and TNF-alpha promoters.

Nuclear factor of activated T cells (NFAT) is a family of transcription factors that regulates activation-induced transcription of many immunologically important genes. Although all NFAT family proteins contain a highly conserved DNA-binding domain and also bind cooperatively with AP-1 proteins to the interleukin-2 (IL-2) promoter NFAT site, each member shows characteristic site preferences to other promoters. Previously, we have shown that NFATc.beta, an isoform of NFATc, is different from NFATp in both DNA binding and transactivation of the TNF-alpha promoter. To further characterize target gene specificity of NFATc and NFATp, we generated deletion mutants as well as mutants swapping the C-terminal region of their DNA binding domains, and analyzed their DNA-binding specificity to different target sites. Our results show that the C-terminal one third of DNA binding domain confers different binding specificity of NFATc and NFATp to an NFAT site in the TNF-alpha promoters. Transient expression of the mutant NFAT proteins also demonstrates that transcriptional activation of the target promoters is consistent with the DNA binding specificity of the mutant NFATs. These results strongly suggest that a binding site preference and availability of different NFAT proteins may program the temporal expression of distinct cytokine genes. Importantly, the C-terminal region of the DNA binding domain plays an important role in determining the binding site preferences at least of NFATp and NFATc members.

RACK-1, a receptor for activated C kinase, interacts with the transcription factor NFAT and represses its transactivation.

To isolate and characterize a novel protein that interacts with nuclear factor of activated T cells (NFAT) and potentially regulates its activity, we screened a Jurkat cDNA library by using the NFAT regulatory domain as bait in the yeast two-hybrid system. RACK-1, a receptor for activated protein kinase C and a homologue of the G-protein beta subunit, was identified as a NFAT-binding protein. Mammalian two hybrid tests in CV-1 cells and a coimmunoprecipitation assay confirmed protein-protein interaction between NFAT and RACK-1. In addition, overexpression of RACK-1 specifically suppressed transcriptional activation derived by NFAT, but not by NF-kappaB. These results demonstrate RACK-1 as a potent negative modulator of NFAT activation and suggest a novel mechanism in NFAT regulation.