The stoichiometry of the human nuclear pore complex is revealed by targeted mass spectrometry and super-resolution microscopy. The analysis reveals that the composition of the nuclear pore and other nuclear protein complexes is remodeled as a function of the cell type.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein–protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Kinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1CENP-U/Q heterodimer, which forms the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1INCENP/Aurora-B core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affected chromosome segregation fidelity in Sli15 wild-type cells. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating CPC-binding and accurate chromosome segregation.

Background: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide (LPS) derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here we utilize a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation. Methods: Acute infection was mimicked by injection of a single dose of LPS into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. Results: LPS treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. LPS treatment led to the deposition of NETs along the arterial lumen and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we employed in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclical peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. Conclusions: Our study shows, that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Alteration of epigenetic modifications plays an important role in human cancer. Notably, the dysregulation of histone post-translational modifications (PTMs) has been associated with several cancers including colorectal cancer (CRC). However, the signature of histone PTMs on circulating nucleosomes is still not well described. We have developed a fast and robust enrichment method to isolate circulating nucleosomes from plasma for further downstream proteomic analysis. This method enabled us to quantify the global alterations of histone PTMs from 9 CRC patients and 9 healthy donors. Among 54 histone proteoforms identified and quantified in plasma samples, 13 histone PTMs were distinctive in CRC. Notably, methylation of histone H3K9 and H3K27, acetylation of histone H3 and citrullination of histone H2A1R3 were upregulated in plasma of CRC patients. A comparative analysis of paired samples identified 3 common histone PTMs in plasma and tumor tissue including the methylation and acetylation state of lysine 27 of histone H3. Moreover, we highlight for the first time that histone H2A1R3 citrulline is a modification upregulated in CRC patients. This new method presented herein allows the detection and quantification of histone variants and histone PTMs from circulating nucleosomes in plasma samples and could be used for biomarker discovery of cancer.