4 research outputs found
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
A Reaction-Based Fluorescent Probe for Imaging of Formaldehyde in Living Cells
Formaldehyde
(FA), in the 0.2–0.4 mM range, is produced
and maintained endogenously via enzymatic pathways. At these levels,
FA can promote cell proliferation as well as mediate memory formation.
Once elevated, FA stress is known to induce cognitive impairments,
memory loss, and neurodegeneration owing to its potent DNA and protein
cross-linking mechanisms. Optical imaging is a powerful noninvasive
approach used to study FA in living systems; however, biocompatible
chemical probes for FA are currently lacking. Herein, we report the
design, synthesis, and biological evaluation of Formaldehyde Probe
1 (FP1), a new fluorescent indicator based on the 2-aza-Cope sigmatropic
rearrangement. The remarkable sensitivity, selectivity, and photostability
of FP1 has enabled us to visualize FA in live HEK293TN and Neuroscreen-1
cells. We envision that FP1 will find widespread applications in the
study of FA associated with normal and pathological processes
Surveillance of Cancer Stem Cell Plasticity Using an Isoform-Selective Fluorescent Probe for Aldehyde Dehydrogenase 1A1
Cancer stem cells
(CSCs) are progenitor cells that contribute to
treatment-resistant phenotypes during relapse. CSCs exist in specific
tissue microenvironments that cell cultures and more complex models
cannot mimic. Therefore, the development of new approaches that can
detect CSCs and report on specific properties (e.g., stem cell plasticity)
in their native environment have profound implications for studying
CSC biology. Herein, we present AlDeSense, a turn-on fluorescent probe
for aldehyde dehydrogenase 1A1 (ALDH1A1) and Ctrl-AlDeSense, a matching
nonresponsive reagent. Although ALDH1A1 contributes to the detoxification
of reactive aldehydes, it is also associated with stemness and is
highly elevated in CSCs. AlDeSense exhibits a 20-fold fluorescent
enhancement when treated with ALDH1A1. Moreover, we established that
AlDeSense is selective against a panel of common ALDH isoforms and
exhibits exquisite chemostability against a collection of biologically
relevant species. Through the application of surface marker antibody
staining, tumorsphere assays, and assessment of tumorigenicity, we
demonstrate that cells exhibiting high AlDeSense signal intensity
have properties of CSCs. Using these probes in tandem, we have identified
CSCs at the cellular level via flow cytometry and confocal imaging,
as well as monitored their states in animal models
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.This work was supported by Goethe University (Corona funds),
the DFG-funded CRC: “Molecular Principles of RNA-Based
Regulation,” DFG infrastructure funds (project numbers:
277478796, 277479031, 392682309, 452632086, 70653611), the
state of Hesse (BMRZ), the Fondazione CR Firenze (CERM),
and the IWB-EFRE-program 20007375. This project has
received funding from the European Union’s Horizon 2020
research and innovation program under Grant Agreement No.
871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the
JQYA at Goethe through project number 2019/AS01. Work in the
lab of KV was supported by a CoRE grant from the University of
New Hampshire. The FLI is a member of the Leibniz Association
(WGL) and financially supported by the Federal Government of
Germany and the State of Thuringia. Work in the lab of RM was
supported by NIH (2R01EY021514) and NSF (DMR-2002837).
BN-B was supported by theNSF GRFP.MCwas supported byNIH
(R25 GM055246 MBRS IMSD), and MS-P was supported by the
HHMI Gilliam Fellowship. Work in the labs of KJ and KT was
supported by Latvian Council of Science Grant No. VPP-COVID
2020/1-0014. Work in the UPAT’s lab was supported by the
INSPIRED (MIS 5002550) project, which is implemented under
the Action “Reinforcement of the Research and Innovation
Infrastructure,” funded by the Operational Program
“Competitiveness, Entrepreneurship and Innovation” (NSRF
2014–2020) and cofinanced by Greece and the EU (European
Regional Development Fund) and the FP7 REGPOT CT-2011-
285950–“SEE-DRUG” project (purchase of UPAT’s 700MHz
NMR equipment). Work in the CM-G lab was supported by
the Helmholtz society. Work in the lab of ABö was supported
by the CNRS, the French National Research Agency (ANR, NMRSCoV2-
ORF8), the Fondation de la Recherche Médicale (FRM,
NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS.
Work in the lab of BM was supported by the Swiss National
Science Foundation (Grant number 200020_188711), the
Günthard Stiftung für Physikalische Chemie, and the ETH
Zurich. Work in the labs of ABö and BM was supported by a
common grant from SNF (grant 31CA30_196256). This work was
supported by the ETHZurich, the grant ETH40 18 1, and the grant
Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble
was supported by the Agence Nationale de Recherche (France)
RA-COVID SARS2NUCLEOPROTEIN and European Research
Council Advanced Grant DynamicAssemblies. Work in the
CA lab was supported by Patto per il Sud della Regione
Siciliana–CheMISt grant (CUP G77B17000110001). Part of
this work used the platforms of the Grenoble Instruct-ERIC
center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the
Grenoble Partnership for Structural Biology (PSB), supported
by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within
the University Grenoble Alpes graduate school (Ecoles
Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-
0003). Work at the UW-Madison was supported by grant
numbers NSF MCB2031269 and NIH/NIAID AI123498. MM
is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of
Science and Innovation (RYC2019-026574-I), and a “La
Caixa” Foundation (ID 100010434) Junior Leader Fellow
(LCR/BQ/PR19/11700003). Funded by project COV20/00764
fromthe Carlos III Institute of Health and the SpanishMinistry
of Science and Innovation to MMand DVL. VDJ was supported
by the Boehringer Ingelheim Fonds. Part of this work used the
resources of the Italian Center of Instruct-ERIC at the CERM/
CIRMMP infrastructure, supported by the Italian Ministry for
University and Research (FOE funding). CF was supported by
the Stiftung Polytechnische Gesellschaft. Work in the lab of
JH was supported by NSF (RAPID 2030601) and NIH
(R01GM123249).Peer reviewe