As part of the Genomics GTL Protein Complex Analysis Project (PCAP) we are developing a high throughput pipeline to purify water soluble protein complexes from D. vulgaris, identify their polypeptide constituents by mass spectrometry, determine their stoichiometries, and provide samples suitable for single particle EM characterization. These methods will then be used as part of PCAP's effort to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites.Our strategy uses a novel "tagless" method that fractionates the water soluble protein contents of a bacterium into a large number of fractions, and then identifies the polypeptide composition of a rational sampling of 10,000-20,000 of these fractions using MALDI TOFITOF mass spectrometry. To establish this method to date: We have developed an optimized four-step fractionation scheme. We have built a prototype multi-channel, native gel electrophoresis instrument for high resolution protein separation and automated band collection that elutes a protein band into a 200 ~l fraction without noticeable loss of sample. We have established an efficient, highly reproducible mass spectrometry sample preparation protocol that uses 96-well PVDF multiscreen plates. We have demonstrated that iTRAQ methodology provides quantitation of the relative abundances of polypeptides in different chromatographic fractions. We have also developed algorithms and graphical display tools for identifying protein complexes from mass spectrometry data, including a method for cluster analysis of iTRAQ data to allow detections of co migrating polypeptides and hence putative protein complexes. Finally, we have refined methods for preparing protein samples suitable for single particle EM analysis. To date, have identified and purified 15 homomeric and heteromeric water soluble protein complexes from D. vulgaris, of whichJive have been sent for EM structural determination, and one of which has been solved at a 17 A resolution. We are further optimizing and refining our methods to establish a fully functional high throughput pipeline.

We introduce an algorithmic framework for tuning the spatial density of disks in a maximal random packing, without changing the sizing function or radii of disks. Starting from any maximal random packing such as a Maximal Poisson-disk Sampling (MPS), we iteratively relocate, inject (add), or eject (remove) disks, using a set of three successively more-aggressive local operations. We may achieve a user-defined density, either more dense or more sparse, almost up to the theoretical structured limits. The tuned samples are conflict-free, retain coverage maximality, and, except in the extremes, retain the blue noise randomness properties of the input. We change the density of the packing one disk at a time, maintaining the minimum disk separation distance and the maximum domain coverage distance required of any maximal packing. These properties are local, and we can handle spatially-varying sizing functions. Using fewer points to satisfy a sizing function improves the efficiency of some applications. We apply the framework to improve the quality of meshes, removing non-obtuse angles; and to more accurately model fiber reinforced polymers for elastic and failure simulations.

Mammalian oocytes are arrested at prophase of the first meiotic division in the primordial follicle pool for months, even years, after birth depending on species, and only a limited number of oocytes resume meiosis, complete maturation, and ovulate with each reproductive cycle. We recently reported that protein phosphatase 6 (PP6), a member of the PP2A-like subfamily, which accounts for cellular serine/threonine phosphatase activity, functions in completing the second meiosis. Here, we generated mutant mice with a specific deletion of Ppp6c in oocytes from the primordial follicle stage by crossing Ppp6cF/F mice with Gdf9-Cre mice and found that Ppp6cF/F; GCre+ mice are infertile. Depletion of PP6c caused folliculogenesis defects and germ cell loss independent of the traditional AKT/mTOR pathway, but due to persistent phosphorylation of H2AX (a marker of double strand breaks), increased susceptibility to DNA damage and defective DNA repair, which led to massive oocyte elimination and eventually premature ovarian failure (POF). Our findings uncover an important role for PP6 as an indispensable guardian of genomic integrity of the lengthy prophase I oocyte arrest, maintenance of primordial follicle pool, and thus female fertility.

An important aim of the Genomics GTL Protein Complex Analysis Project (PCAP) is the isolation and identification of membrane protein complexes from D. vulgaris. The cataloging of complex subunit constituents from this organism, grown under normal and stressed conditions, will support the long-range goal of modeling stress responses in D. vulgaris relevant to the detoxification of metal and radionuclide contaminated sites. Isolation of endogenous D. vulgaris membrane protein complexes in quantities sufficient for chromatographic analysis requires substantial amounts of cell membranes. Twenty liter cultures of D. vulgaris typically yielded about 50 milligrams of total membrane proteins. To extract inner and outer membrane protein complexes stably and with maximal yield, we have used a multi-step procedure in which the bacterial membrane is sequentially processed. Mernbranes were initially treated with a mild detergent to extract proteins primarily from the inner membrane. The residual membrane pellet, enriched with proteins of the outer membrane, was solubilized using a more aggressive detergent. Chromatographic procedures found effective in purifying complexes involved the use of ion exchange, hydroxyapatite and molecular sieve media. SDS-PAGE of the various chromatographic fractions was used to tentatively identify subunits of complexes based on a display of co-elution. Molecular sieve fractions were subjected to native PAGE to further isolate potential complexes. Proteins of the native gel bands, in tum, were extracted and run on SDSPAGE. These gels revealed whether a putative complex was indeed composed of lower molecular weight subunits, and provided samples well suited for in-gel processing and mass spectrometry analysis. In this manner, a number of homo-and heteromeric complexes, ranging in weight from about 70 to 400 kDa, have been identified in this first year. Use of a free-flow electrophoresis device being developed in our group in combination with larger-scale processing of cell membranes is expected to significantly improve the isolation and identification of complexes over the next project year.

Study question

There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis.

Summary answer

In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion.

What is known already

N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies.

Study design, samples/materials, methods

By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test.

Main results and the role of chance

Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P < 0.001 compared to control). Further analysis showed that a defective midbody may be responsible for the failure of cytokinesis completion.

Limitations, reasons for caution

The present study did not include a detailed analysis of the mechanisms underlying the results, which will require more extensive further investigations.

Wider implications of the findings

N-WASP may play an important role in mediating and co-ordinating the activity of the spindle (midbody) and actin (contractile ring constriction) when cell division occurs. The findings are important for understanding the regulation of oocyte meiosis completion and failures in this process that affect oocyte quality.

Large scale data

None.

Study funding and competing interests

This work was supported by the National Basic Research Program of China (No. 2012CB944404) and the National Natural Science Foundation of China (Nos 30930065, 31371451, 31272260 and 31530049). There are no potential conflicts of interests.

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

Unlabelled

Semen enhances HIV infection in vitro, but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity.

Importance

Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro, but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen.