SUMMARY Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal, in a process that requires PARP activity. Whether regressed forks can efficiently restart and the factors required to restart fork progression after fork reversal are still unknown. Here we combined biochemical and electron microscopy approaches with single-molecule DNA fiber analysis, to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition, not shared by other human RecQ proteins. We show that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

Summary The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA binding protein SAM68 which associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.

The AAA-ATPase VCP/p97 cooperates with distinct cofactors to process ubiquitinated proteins in different cellular pathways 1–3. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear 4, 5. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds the plasma membrane protein caveolin-1 (Cav1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitinated Cav1 in SDS-resistant high molecular weight complexes on endosomes, which are en route to degradation in endolysosomes 6. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of Cav1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific Caveolin-3 (Cav3) accumulates in sarcoplasmic pools and specifically delocalises from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitinated cargo in the endocytic pathway and suggest that impaired trafficking of caveolin may contribute to the pathogenesis in individuals with VCP mutations.

The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.