SummaryDuring interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.

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Highlights d Global quantification of assembly state changes in the mitotic proteome d Improved performance over thermostability measurement of proteome states d Discovery of a mitotic disassembly intermediate of the nuclear pore complex d Introduction of SECexplorer-cc, a publicly available online platform

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K D ‫؍‬ 1.3 ؋ 10 ؊6 M). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.A large body of evidence now shows that macrophage migration inhibitory factor (MIF) 2 activates a range of intracellular pathways and plays a key role in host immune and inflammatory responses (1, 2). Certain of the MIF inflammatory functions also have been proposed to be the result of the unusual enzymatic properties of the protein, namely tautomerase and oxidoreductase activities (3-6). Inhibition or deletion of MIF attenuates disease progression in experimental models such as atherosclerosis, arthritis, glomerulonephritis, sepsis, autoimmune encephalitis, and autoimmune diabetes (7-13). A pivotal step in the inflammatory response is the chemokine-governed adherence of monocytes to the endothelial lining which is then followed by their egress from the vasculature at the affected site. Earlier data from MIF Ϫ/Ϫ mice illustrate a role of MIF in leukocyte recruitment that was recently substantiated by the finding that MIF serves as a chemoattractant for monocytes and T cells by directly binding to the chemokine receptors CXCR2 and CXCR4 (14, 15). On the cell surface MIF also associates with CD74 (invariant chain of major histocompatibility complex class II) which colocalizes with CXCR2 (14, 16). Interaction with different surface molecules is thought to partly explain the wide impact of MIF on cellular pathways.Despite its role as a key mediator in immune and inflammatory diseases, very little is known of how MIF action is regulated and terminated. Accordingly, we searched for endogenous molecules with the ability to control key steps of MIF signaling (i.e. receptor binding and/or receptor-associated functions). In this study, we identified ribosomal protein S19 (RPS19), a component of the small...

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in-bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non-invasive three-dimensional (3D) imaging technology for in vivo monitoring, termed flat-panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high-resolution fpvCT. A proteomics approach using 2D SDS-PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER-60, alpha-1-anti-trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF-2), and phosphoglycerate kinase 1. Hsp70, ODF-2, hnRNP H1, and ER-60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory-type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox-62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre-immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF-2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation-based male infertility.