SUMMARY53BP1 is a multi-functional double-strand break (DSB) repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumors to PARP inhibitors. Central to all 53BP1 activities is its recruitment to DSBs via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, TIRR (Tudor Interacting Repair Regulator) that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, ATM phosphorylates 53BP1 and recruits RIF1 to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to DSBs. Depletion of TIRR destabilizes 53BP1 in the nuclear soluble fraction and also alters the DSB-induced protein complex centering 53BP1. These findings identify TIRR as a new factor that influences DSB †

Background: Malaria pathogenesis depends on intricate functioning of ER secretory pathway; however, it lacks canonical UPR machinery to respond to ER stress. Results: Malaria parasite fails to induce ER chaperones and converts to gametocytes upon ER stress. Conclusion: Malaria parasite escapes ER stress by switching to the sexual stage. Significance: ER stress may serve as a physiological cue for parasites to undergo gametocytogenesis.

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer’s cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.