Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies

RENEB accident simulation exercise

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested

First detection of X-ray polarization from the accreting neutron star 4U 1820-303

This paper reports the first detection of polarization in the X-rays for atoll-source 4U 1820-303, obtained with the Imaging X-ray Polarimetry Explorer (IXPE) at 99.999% confidence level (CL). Simultaneous polarimetric measurements were also performed in the radio with the Australia Telescope Compact Array (ATCA). The IXPE observations of 4U 1820-303 were coordinated with Swift-XRT, NICER, and NuSTAR aiming to obtain an accurate X-ray spectral model covering a broad energy interval. The source shows a significant polarization above 4 keV, with a polarization degree of 2.0(0.5)% and a polarization angle of -55(7) deg in the 4-7 keV energy range, and a polarization degree of 10(2)% and a polarization angle of -67(7) deg in the 7-8 keV energy bin. This polarization also shows a clear energy trend with polarization degree increasing with energy and a hint for a position-angle change of about 90 deg at 96% CL around 4 keV. The spectro-polarimetric fit indicates that the accretion disk is polarized orthogonally to the hard spectral component, which is presumably produced in the boundary/spreading layer. We do not detect linear polarization from the radio counterpart, with a 99.97% upper limit of 50% at 7.25 GHz

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Solution structure of recombinant Pvfp-5β reveals insights into mussel adhesion

Solution structure of byssal plaque protein Pvfp-5 beta secreted by the Asian green mussel Perna viridis gives molecular insight into mussel adhesion on wet surfaces.Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5 beta, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5 beta has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the mu s-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5 beta is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials

Backbone chemical shift spectral assignments of SARS coronavirus-2 non-structural protein nsp9

As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

<p>The Data Set is a collection of the Fasta of sequencing of 6 human library of scFvs (VH-linker VL) obtain from the IgM repertoire and of 6 human libraries of scFvs (VH-linker VL) obtain from the IgG_IgA repertoire of 6 patients that recover from COVID19.</p> <p>Each of the 12 libraries (6 for the IgM and 6 for the IaG/IgA repertoire) were sequenced by ION TORRENT technology by Genomnia. For each scFv library an independently VH and a VL Ion Torrent library was constructed as follows. VH and VL were amplified using primers (VH_fus_F- VH_fus_R and VL_fus_F - VL_fus_R_BXX) having, at the 3’end, the region presents in the pLinker220 plasmid flanking the VH or VL and, at the 5’ end, the Ion Torrent adaptors. In this step, Unique Molecular Identifiers (UMI) consisting of 13 degenerate bases are also introduced in the reverse primer used to amplify the VH and VH regions.  The amplified VH and VL were then subjected to a second PCR using the primers A_fus – trP1_fus to build the fusion library. Sequencing was performed on an Ion Torrent platform (IonS5) with 400bp chemistry on a 530 chip. Both types of sequencing libraries were constructed by Genomnia. Each VH library was individually sequenced to a 400bp read on a 530 chip, yielding approximately 20-25 million reads per sample. The VL libraries, on the other hand, were barcoded and sequenced in a pool of three libraries from different patients, aiming to obtain 6-10 million reads per library. All sequencing runs were of good quality, with an average of 73% of the reads mapping to immunoglobulin genes used for clonotype identification (i.e. less than 30% polyclonal reads) and less than 20% low-quality sequences</p> <p> </p> <p><span> The correspondence between the library number (#) and the # of the patient in the sequencing  files name is as follows:</span></p> <table> <tbody> <tr> <td> <p><strong><span>Library #</span></strong></p> </td> <td> <p><strong><span>Patient # in the file name</span></strong></p> </td> </tr> <tr> <td> <p><span>lib 1 </span></p> </td> <td> <p><span>pz24</span></p> </td> </tr> <tr> <td> <p><span>lib 2 </span></p> </td> <td> <p><span>pz26</span></p> </td> </tr> <tr> <td> <p><span>lib 3 </span></p> </td> <td> <p><span>pz27</span></p> </td> </tr> <tr> <td> <p><span>lib 4 </span></p> </td> <td> <p><span>pz39</span></p> </td> </tr> <tr> <td> <p><span>lib 5 </span></p> </td> <td> <p><span>pz42</span></p> </td> </tr> <tr> <td> <p><span>lib 6 </span></p> </td> <td> <p><span>pz44</span></p> </td> </tr> </tbody> </table> <p><span> </span></p> <p> </p> <p>The correspondence between the library number (#) and the file name for each of the VH and  sequencing is as follows:</p> <table> <tbody> <tr> <td> <p><strong><span>Library #</span></strong></p> </td> <td> <p><strong><span>File name VH Sequences</span></strong></p> </td> </tr> <tr> <td> <p><span>lib 1 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-163-30pM_24_IgM_VH_414__REANALYSIS_488_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 1 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-162-30pM_24_IgG_IgA_VH_407_469_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 2 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-157-30pM_pz26_VH_IgM_404_REANALYSIS_460_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 2 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-158-30pM_pz26_VH_IgG_IgA_403_458_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 3 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-161-30pM_27_IgM_VH_408_465_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 3 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-164-30pM_27_IgG_IgA_VH_413_482_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 4 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_II_Auto_user_S5-00513-159-30pM_pz39_IgM_VH_406_461_rawtf.fa</span></p> <p><span>PANANTICOVID_I_Auto_user_S5-00513-159-30pM_pz39_IgM_VH_406_REANALYSIS_477_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 4 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-160-30pM_pz39_IgG_IgA_VH_405_463_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 5 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_II_Auto_user_S5-00513-166-30pM_pz42_VH_IgM_415_486_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 5 IgG_IgA VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-167-30pM_42_IgG_IgA_VH_418_489_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 6 IgM VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-168-30pM_44_IgM_VH_417_491_rawtf.fa</span></p> </td> </tr> <tr> <td> <p><span>lib 6 IgG_IgA<span>  </span>VH</span></p> </td> <td> <p><span>PANANTICOVID_I_Auto_user_S5-00513-169-30pM_pz44_IgG_IgA_VH_420_493_rawtf.fa</span></p> </td> </tr> </tbody> </table&gt

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

Abstract In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications

The Instrument of the Imaging X-Ray Polarimetry Explorer

While X-ray spectroscopy, timing, and imaging have improved much since 1962 when the first astronomical nonsolar source was discovered, especially wi the launch of the Newton/X-ray Multi-Mirror Mission, Rossi/X-ray Timing Explorer, and Chandra/Advanced X-ray Astrophysics Facility, the progress of X-ray polarimetry has been meager. This is in part due to the lack of sensitive polarization detectors, which in turn is a result of the fate of approved missions and because celestial X-ray sources appear less polarized than expected. Only one positive measurement has been available until now: the Orbiting Solar Observatory measured the polarization of the Crab Nebula in the 1970s. The advent of microelectronics techniques has allowed for designing a detector based on the photoelectric effect of gas in an energy range where the optics are efficient at focusing in X-rays. Here we describe the instrument, which is the major contribution of the Italian collaboration to the Small Explorer mission called IXPE, the Imaging X-ray Polarimetry Explorer, which will launch in late 2021. The instrument is composed of three detector units based on this technique and a detector service unit. Three mirror modules provided by Marshall Space Flight Center focus X-rays onto the detectors. We show the technological choices, their scientific motivation, and results from the calibration of the instrument. IXPE will perform imaging, timing, and energy-resolved polarimetry in the 2–8 keV energy band opening this window of X-ray astronomy to tens of celestial sources of almost all classes