Investigating Posttranslational Modifications of Hsp90 and the Epichaperome in Embryonic Stem Cells

Heat shock protein 90 (Hsp90) is a highly conserved and essential molecular chaperone in eukaryotes. Through interactions with various co-chaperones, Hsp90 is able to adapt to meet the specific needs of a structurally and functionally diverse, but specific set of clienteles. The diversity of its clients makes it a key regulator of nearly all cellular processes, and it serves as a central hub for many different signaling networks. One of the major functions of the Hsp90 chaperone machinery is in the modulation of protein homeostasis, a protective function often exploited in cancer cells in order to buffer oncogenic mutations and the overexpression of numerous oncoproteins, thus linking Hsp90 to tumorigenesis. Inhibitors of Hsp90 have entered clinical trials displaying distinct pharmacological efficacies and toxicological profiles. In this study we first investigate the posttranslational and conformational states of Hsp90 targeted by either PU-H71 or geldanamycin (GA) using mass spectrometry. Quantitative analysis suggests that PU-H71 binds to Hsp90 in a multi-chaperone complex with an adopted closed-like conformation, while GA binds to uncomplexed Hsp90 in an open-like conformation. Additionally, phosphorylation was found to be enriched in the acidic linker region of Hsp90 targeted by PU-H71. Mutational analysis revealed that phosphorylation of S226/S255 promotes association of Hsp90 with epichaperome related cochaperones (Cdc37, FKBP4, and Aha1). Further investigation revealed that S255 phosphorylation was particularly important for promoting ESC self-renewal providing a possible biochemical mechanism by which epichaperome formation is promoted and linked to the maintenance of ESCs. In order to expand the scope of our analysis when evaluating Hsp90 complexes and the PTMs that regulate them we then developed a novel method for purification and separation of Hsp90 complexes prior to MS-based proteomics analysis. This new method involved expression of an affinity tagged Hsp90 containing a recognizable proteolytic sequence, which allows capture and separation of Hsp90 complexes under native conditions. We demonstrate that this technique we termed as a native elution allows for detailed characterization of Hsp90 complexes, previously unattainable. Co-chaperones and clients acquire distinct distribution profiles based off the Hsp90 complexes they associate with. Furthermore, subunits of large protein complexes appear with very distinctive distribution profiles generally with significant enrichment in only a few of the high-molecular weight species. Lastly, we identified multiple Hsp90 PTMs across various Hsp90 complexes. As PTMs remain an important determinant of Hsp90 inhibitor sensitivity, the quantitative data will be used for continued bioinformatic analyses of these modifications, in which a functional relationship between modification(s) and Hsp90-bound proteins will be mapped

Neptune Odyssey: A Flagship Concept for the Exploration of the Neptune–Triton System

The Neptune Odyssey mission concept is a Flagship-class orbiter and atmospheric probe to the Neptune-Triton system. This bold mission of exploration would orbit an ice-giant planet to study the planet, its rings, small satellites, space environment, and the planet-sized moon Triton. Triton is a captured dwarf planet from the Kuiper Belt, twin of Pluto, and likely ocean world. Odyssey addresses Neptune system-level science, with equal priorities placed on Neptune, its rings, moons, space environment, and Triton. Between Uranus and Neptune, the latter is unique in providing simultaneous access to both an ice giant and a Kuiper Belt dwarf planet. The spacecraft - in a class equivalent to the NASA/ESA/ASI Cassini spacecraft - would launch by 2031 on a Space Launch System or equivalent launch vehicle and utilize a Jupiter gravity assist for a 12 yr cruise to Neptune and a 4 yr prime orbital mission; alternatively a launch after 2031 would have a 16 yr direct-to-Neptune cruise phase. Our solution provides annual launch opportunities and allows for an easy upgrade to the shorter (12 yr) cruise. Odyssey would orbit Neptune retrograde (prograde with respect to Triton), using the moon's gravity to shape the orbital tour and allow coverage of Triton, Neptune, and the space environment. The atmospheric entry probe would descend in ~37 minutes to the 10 bar pressure level in Neptune's atmosphere just before Odyssey's orbit-insertion engine burn. Odyssey's mission would end by conducting a Cassini-like "Grand Finale,"passing inside the rings and ultimately taking a final great plunge into Neptune's atmosphere

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

Catalog of Earthquake Hypocenters at Alaskan Volcanoes: January 1, 2000 through December 31, 2001

The Alaska Volcano Observatory (AVO), a cooperative program of the U.S. Geological Survey, the Geophysical Institute of the University of Alaska Fairbanks, and the Alaska Division of Geological and Geophysical Surveys, has maintained seismic monitoring networks at potentially active volcanoes in Alaska since 1988 (Power and others, 1993; Jolly and others, 1996; Jolly and others, 2001). The primary objectives of this program are the seismic surveillance of active, potentially hazardous, Alaskan volcanoes and the investigation of seismic processes associated with active volcanism. This catalog reflects the status and evolution of the seismic monitoring program, and presents the basic seismic data for the time period January 1, 2000, through December 31, 2001. For an interpretation of these data and previously recorded data, the reader should refer to several recent articles on volcano related seismicity on Alaskan volcanoes in Appendix G. The AVO seismic network was used to monitor twenty-three volcanoes in real time in 2000-2001. These include Mount Wrangell, Mount Spurr, Redoubt Volcano, Iliamna Volcano, Augustine Volcano, Katmai Volcanic Group (Snowy Mountain, Mount Griggs, Mount Katmai, Novarupta, Trident Volcano, Mount Mageik, Mount Martin), Aniakchak Crater, Pavlof Volcano, Mount Dutton, Isanotski Peaks, Shishaldin Volcano, Fisher Caldera, Westdahl Peak, Akutan Peak, Makushin Volcano, Great Sitkin Volcano, and Kanaga Volcano (Figure 1). AVO located 1551 and 1428 earthquakes in 2000 and 2001, respectively, on and around these volcanoes. Highlights of the catalog period (Table 1) include: volcanogenic seismic swarms at Shishaldin Volcano between January and February 2000 and between May and June 2000; an eruption at Mount Cleveland between February and May 2001; episodes of possible tremor at Makushin Volcano starting March 2001 and continuing through 2001, and two earthquake swarms at Great Sitkin Volcano in 2001. This catalog includes: (1) earthquake origin times, hypocenters, and magnitudes with summary statistics describing the earthquake location quality; (2) a description of instruments deployed in the field and their locations; (3) a description of earthquake detection, recording, analysis, and data archival systems; (4) station parameters and velocity models used for earthquake locations; (5) a summary of daily station usage throughout the catalog period; and (6) all HYPOELLIPSE files used to determine the earthquake locations presented in this report

Catalog of Earthquake Hypocenters at Alaskan Volcanoes: January 1 through December 31, 2003

The Alaska Volcano Observatory (AVO), a cooperative program of the U.S. Geological Survey, the Geophysical Institute of the University of Alaska Fairbanks, and the Alaska Division of Geological and Geophysical Surveys, has maintained seismic monitoring networks at historically active volcanoes in Alaska since 1988 (Power and others, 1993; Jolly and others, 1996; Jolly and others, 2001; Dixon and others, 2002). The primary objectives of this program are the seismic monitoring of active, potentially hazardous, Alaskan volcanoes and the investigation of seismic processes associated with active volcanism. This catalog presents the basic seismic data and changes in the seismic monitoring program for the period January 1, 2002 through December 31, 2002. Appendix G contains a list of publications pertaining to seismicity of Alaskan volcanoes based on these and previously recorded data. The AVO seismic network was used to monitor twenty-four volcanoes in real time in 2002. These include Mount Wrangell, Mount Spurr, Redoubt Volcano, Iliamna Volcano, Augustine Volcano, Katmai Volcanic Group (Snowy Mountain, Mount Griggs, Mount Katmai, Novarupta, Trident Volcano, Mount Mageik, Mount Martin), Aniakchak Crater, Mount Veniaminof, Pavlof Volcano, Mount Dutton, Isanotski Peaks, Shishaldin Volcano, Fisher Caldera, Westdahl Peak, Akutan Peak, Makushin Volcano, Great Sitkin Volcano, and Kanaga Volcano (Figure 1). Monitoring highlights in 2002 include an earthquake swarm at Great Sitkin Volcano in May-June; an earthquake swarm near Snowy Mountain in July-September; low frequency (1-3 Hz) tremor and long-period events at Mount Veniaminof in September-October and in December; and continuing volcanogenic seismic swarms at Shishaldin Volcano throughout the year. Instrumentation and data acquisition highlights in 2002 were the installation of a subnetwork on Okmok Volcano, the establishment of telemetry for the Mount Veniaminof subnetwork, and the change in the data acquisition system to an EARTHWORM detection system. AVO located 7430 earthquakes during 2002 in the vicinity of the monitored volcanoes. This catalog includes: (1) a description of instruments deployed in the field and their locations; (2) a description of earthquake detection, recording, analysis, and data archival systems; (3) a description of velocity models used for earthquake locations; (4) a summary of earthquakes located in 2002; and (5) an accompanying UNIX tar-file with a summary of earthquake origin times, hypocenters, magnitudes, and location quality statistics; daily station usage statistics; and all HYPOELLIPSE files used to determine the earthquake locations in 2002. The AVO seismic network was used to monitor twenty-four volcanoes in real time in 2002. These include Mount Wrangell, Mount Spurr, Redoubt Volcano, Iliamna Volcano, Augustine Volcano, Katmai Volcanic Group (Snowy Mountain, Mount Griggs, Mount Katmai, Novarupta, Trident Volcano, Mount Mageik, Mount Martin), Aniakchak Crater, Mount Veniaminof, Pavlof Volcano, Mount Dutton, Isanotski Peaks, Shishaldin Volcano, Fisher Caldera, Westdahl Peak, Akutan Peak, Makushin Volcano, Great Sitkin Volcano, and Kanaga Volcano (Figure 1). Monitoring highlights in 2002 include an earthquake swarm at Great Sitkin Volcano in May-June; an earthquake swarm near Snowy Mountain in July-September; low frequency (1-3 Hz) tremor and long-period events at Mount Veniaminof in September-October and in December; and continuing volcanogenic seismic swarms at Shishaldin Volcano throughout the year. Instrumentation and data acquisition highlights in 2002 were the installation of a subnetwork on Okmok Volcano, the establishment of telemetry for the Mount Veniaminof subnetwork, and the change in the data acquisition system to an EARTHWORM detection system. AVO located 7430 earthquakes during 2002 in the vicinity of the monitored volcanoes. This catalog includes: (1) a description of instruments deployed in the field and their locations; (2) a description of earthquake detection, recording, analysis, and data archival systems; (3) a description of velocity models used for earthquake locations; (4) a summary of earthquakes located in 2002; and (5) an accompanying UNIX tar-file with a summary of earthquake origin times, hypocenters, magnitudes, and location quality statistics; daily station usage statistics; and all HYPOELLIPSE files used to determine the earthquake locations in 2002