Aurora B-dependent Ndc80 degradation regulates kinetochore composition in meiosis.

The kinetochore complex is a conserved machinery that connects chromosomes to spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore remodeling is mediated by the temporal changes in the abundance of a single subunit called Ndc80. We previously described the regulatory events that control the timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has been previously implicated in correcting erroneous microtubule-kinetochore attachments, is essential for Ndc80 degradation in a microtubule-independent manner. The N terminus of Ndc80, including a 27-residue sequence and Aurora B phosphorylation sites, is both necessary and sufficient for kinetochore protein degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate their kinetochore composition through regulated Ndc80 degradation, and demonstrates that Aurora B-dependent regulation of kinetochores extends beyond altering microtubule attachments.

Proteomic analysis of bronchoalveolar lavage fluid in rat exposed to TiO2 nanostructured aerosol by inhalation.

The pulmonary toxicological properties of inhaled titanium dioxide were studied using bronchoalveolar lavage fluid (BALF) cytology and proteomics analyses. Fischer 344 rats were exposed to 10 mg/m3 of TiO2 nanostructured aerosol by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. As previously described, cytological analyses of BALF showed a strong inflammatory response up to 3 post-exposure days, which persisted however, at a lower intensity up to 180 days. In addition, using Multidimensional Protein Identification Technology (MudPIT), we identified a total of 107, 50 and 45 proteins (UniprotKB identifiers) differentially expressed in exposed rats immediately, 3 and 180 days after the end of exposure respectively. Increased levels of inflammatory proteins, members of proteasome, various histones, proteins involved in cytoskeleton organization, were noticed up to 3 days (short-term response). Some of these proteins were linked with Neutrophil Extracellular Trap formation (NETosis). Long-term response was also characterized by a persistent altered expression of proteins up to 180 days. Altogether, these results suggest that exposure to low toxicity low solubility nanomaterials such as TiO2 may induce long-term changes in the pulmonary protein expression pattern of which the physio-pathological consequences are unknown. SIGNIFICANCE: This paper describes in rats, at the pulmonary level, the effects of inhaled nanostructured aerosol of TiO2 on the secreted proteins found in the broncho-alveolar space by comparing the proteomic profile in broncho-alveolar lavage fluid supernatants of control and exposed animals. This work brings new insights about the early events occurring following the end of exposure and suggests the formation of Neutrophil Extracellular Traps (NETosis) that could be interpret as a potential early mechanism of defense against TiO2 nanoparticles. This work also describes the long term effects (180 post-exposure days) of such an exposure and the change in secreted protein expression in the absence of significant histopathological modifications.

Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer.

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5' exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5' exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.

The Drosophila splicing factor PSI is phosphorylated by casein kinase II and tousled-like kinase.

Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the Drosophila splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in Drosophila S2 tissue culture cells, the majority of which are splicing factors.

Cerebrospinal fluid-based kinetic biomarkers of axonal transport in monitoring neurodegeneration.

Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid-based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water (2H2O), distinct, newly synthesized 2H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2H2O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson's disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.

Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1.

Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins, and numerous associated "nonribosomal" proteins. To identify nonribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, glycogen synthase 1 (GYS1), was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, by virtue of its ribosomal association, provides a feedback loop between the energy state of the cells and the translation machinery.

Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome.

The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.

Humanization of an anti-ICAM-1 antibody with over 50-fold affinity and functional improvement.

The monoclonal antibody 1A6 binds to human intercellular adhesion molecule 1 (ICAM-1, CD54) and inhibits infection by 90% of human rhinovirus (HRV) serotypes. To make a therapeutic molecule for preventing and treating HRV infection, we humanized a single chain antibody (scFv), 1A6, by a structure-guided complementarity-determining region (CDR) grafting procedure. Our final humanized 1A6 scFv does not retain any mouse back mutations in the framework. Without changing the CDR sequences, the humanized 1A6 scFv demonstrates over 50-fold improvement in both affinity for ICAM-1 and protection efficacy against HRV infection in vitro.

Prevention of human rhinovirus infection by multivalent fab molecules directed against ICAM-1.

We have developed a technology for improving avidity by making bivalent, trivalent, or tetravalent recombinant polypeptides. We designed tripartite proteins consisting of the Fab fragment of an antibody fused with a hinge derived from human immunoglobulin D that was further linked to polymerization domains derived from human coiled-coil proteins. We report here on the application of this method with a Fab domain directed against the major human rhinovirus receptor, intercellular adhesion molecule 1 (ICAM-1). Multivalent anti-ICAM-1 molecules were produced in bacteria and purified as soluble preassembled homogeneous proteins at high yield. These proteins successfully blocked rhinovirus infection in vitro, with the efficiency increasing from monomer to dimer, trimer, and tetramer. The diminished dissociation rate of these multivalent antibodies and their improved efficacy in preventing rhinovirus infection provide a foundation for producing prophylactic and therapeutic molecules against human rhinovirus, the causative agent of the majority of common colds.