Marie-Claude Landry

53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone ‘code’ produced by DSB signalling.

The ubiquitous role of ubiquitin in the DNA damage response.

Abstract Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response. In this review, we discuss how DNA repair and checkpoint pathways utilize the diversity offered by the ubiquitin conjugation system to modulate the response to genotoxic lesions in space and time. In particular, we will highlight recent work done on the regulation of DNA double-strand breaks signalling and repair by the RNF8/RNF168 E3 ubiquitin ligases, the Fanconi anemia pathway and the role of protein degradation in the enforcement and termination of checkpoint signalling. We also discuss the various functions of deubiquitylating enzymes in these processes along with potential avenues for exploiting the ubiquitin conjugation/deconjugation system for therapeutic purposes.

A strategy for modulation of enzymes in the ubiquitin system.

Modifying Deubiquitinases Protein ubiquitination is a widespread mechanism for cellular regulation, and new regulators are valuable research tools and may help to generate therapeutic small molecules. Ernst et al. (p. 590, published online 3 January) used known crystal structures to roughly define the interaction domain between a ubiquitin-specific protease and a ubiquitinated substrate and then screened ubiquitin variants with changes in these residues to find variants that acted as potent and specific regulators that could modify ubiquitin pathway regulation in cells. A technique for developing specific and potent enzyme inhibitors is validated on enzymes of the ubiquitin‑proteasome system. The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

Regulation of Cell Death by Recycling Endosomes and Golgi Membrane Dynamics via a Pathway Involving Src-family kinases, Cdc42 and Rab11a

Actin dynamics and membrane trafficking influence cell commitment to programmed cell death through largely undefined mechanisms. To investigate how actin and recycling endosome (RE) trafficking can engage death signaling, we studied the death program induced by the adenovirus early region 4 open reading frame 4 (E4orf4) protein as a model. We found that in the early stages of E4orf4 expression, Src-family kinases (SFKs), Cdc42, and actin perturbed the organization of the endocytic recycling compartment and promoted the transport of REs to the Golgi apparatus, while inhibiting recycling of protein cargos to the plasma membrane. The resulting changes in Golgi membrane dynamics that relied on actin-regulated Rab11a membrane trafficking triggered scattering of Golgi membranes and contributed to the progression of cell death. A similar mobilization of RE traffic mediated by SFKs, Cdc42 and Rab11a also contributed to Golgi fragmentation and to cell death progression in response to staurosporine, in a caspase-independent manner. Collectively, these novel findings suggest that diversion of RE trafficking to the Golgi complex through a pathway involving SFKs, Cdc42, and Rab11a plays a general role in death signaling by mediating regulated changes in Golgi dynamics.

A Functional Interplay between the Small GTPase Rab11a and Mitochondria-Shaping Proteins Regulates Mitochondrial Positioning and Polarization of the Actin Cytoskeleton Downstream of Src-Family Kinases

Background: Mitochondrial dynamics are integrated within signaling systems through ill-defined mechanisms. Results: During cytoskeletal rearrangements by Src family kinases (SFK), Rab11a modulates mitochondrial dynamics that, in turn, influence actin assembly. Conclusion: Redistribution of mitochondria near actin-rich structures is mediated by SFK and Rab11a and facilitates polarization of the cytoskeleton. Significance: A new functional connection is uncovered between membrane traffic and mitochondrial dynamics during cellular remodeling. It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

Src-family kinase signaling, actin-mediated membrane trafficking and organellar dynamics in the control of cell fate: Lessons to be learned from the adenovirus E4orf4 death factor

Evidence has accumulated that there are different modes of regulated cell death, which share overlapping signaling pathways. Cytoskeletal-dependent inter-organellar communication as a result of protein and lipid trafficking in and out of organelles has emerged as a common, key issue in the regulation of cell death modalities. The movement of proteins and lipids between cell compartments is believed to relay death signals in part through modifications of organelles dynamics. Little is known, however, regarding how trafficking is integrated within stress signaling pathways directing organelle-specific remodeling events. In this review, we discuss emerging evidence supporting a role for regulated changes in actin dynamics and intracellular membrane flow. Based on recent findings using the adenovirus E4orf4 death factor as a probing tool to tackle the mechanistic underpinnings that control alternative modes of cell death, we propose the existence of multifunctional platforms at the endosome-Golgi interface regulated by SFK-signaling. These endosomal platforms could be mobilized during cell activation processes to reorganize cellular membranes and promote inter-organelle signaling.