Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

Background: Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB). Results: In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD(75) tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD(28). Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3' position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1' position from I-29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP 2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5' leader sequence (5'UTR) of BNIP-2. Conclusions: Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid

Simple peptide quantification approach for MS-based proteomics quality control

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 mu L of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC-MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 mu g as an optimal peptide amount to be injected into this particular LC-MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC-MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control

The Online Protein Processing Resource (TOPPR) : a database and analysis platform for protein processing events

We here present The Online Protein Processing Resource (TOPPR; http://iomics.ugent.be/toppr/), an online database that contains thousands of published proteolytically processed sites in human and mouse proteins. These cleavage events were identified with COmbinded FRActional DIagonal Chromatography proteomics technologies, and the resulting database is provided with full data provenance. Indeed, TOPPR provides an interactive visual display of the actual fragmentation mass spectrum that led to each identification of a reported processed site, complete with fragment ion annotations and search engine scores. Apart from warehousing and disseminating these data in an intuitive manner, TOPPR also provides an online analysis platform, including methods to analyze protease specificity and substrate-centric analyses. Concretely, TOPPR supports three ways to retrieve data: (i) the retrieval of all substrates for one or more cellular stimuli or assays; (ii) a substrate search by UniProtKB/Swiss-Prot accession number, entry name or description; and (iii) a motif search that retrieves substrates matching a user-defined protease specificity profile. The analysis of the substrates is supported through the presence of a variety of annotations, including predicted secondary structure, known domains and experimentally obtained 3D structure where available. Across substrates, substrate orthologs and conserved sequence stretches can also be shown, with iceLogo visualization provided for the latter

The Far Side of Mars: Two Distant Marsquakes Detected by InSight

For over three Earth years the Marsquake Service has been analyzing the data sent back from the Seismic Experiment for Interior Structure¿the seismometer placed on the surface of Mars by NASA¿s InSight lander. Although by October 2021, the Mars seismic catalog included 951 events, until recently all these events have been assessed as lying within a radius of 100° of InSight. Here we report two distant events that occurred within days of each other, located on the far side of Mars, giving us our first glimpse into Mars¿ core shadow zone. The first event, recorded on 25 August 2021 (InSight sol 976), shows clear polarized arrivals that we interpret to be PP and SS phases at low frequencies and locates to Valles Marineris, 146° ± 7° from InSight. The second event, occurring on 18 September 2021 (sol 1000), has significantly more broadband energy with emergent PP and SS arrivals, and a weak phase arriving before PP that we interpret as Pdiff¿. Considering uncertain pick times and poorly constrained travel times for Pdiff¿, we estimate this event is at a distance between 107° and 147° from InSight. With magnitudes of MMaw 4.2 and 4.1, respectively, these are the largest seismic events recorded so far on Mars.Anna C. Horleston, Jessica C. E. Irving,and Nicholas A. Teanby are funded by the UKSA under Grant Numbers ST/R002096/1, ST/W002523/1, and ST/W002515/1.Nikolaj L. Dahmen, Cecilia Duran, Géraldine Zenhäusern, andSimon C. Stähler would like to acknowledge support from Eidgenössische Technische Hochschule (ETH) through the ETH+ funding scheme (ETH+02 19-1: “Planet Mars”). The French coauthors acknowledge the funding support provided by CNES and the Agence Nationale de la Recherche (ANR-19-CE31-0008-08 MAGIS) for SEIS operation and SEIS Science analysis. Alexander E. Stott acknowledges the French Space Agency CNES and ANR (ANR-19-CE31-0008-08). Caroline Beghein and Jiaqi Li were supported by NASA InSight Participating Scientist Program (PSP) Grant Number 80NSSC18K1679. This article is InSight Contribution Number 236

Seismic Constraints on the Thickness and Structure of the Martian Crust from InSight

NASA¿s InSight mission [1] has for the first time placed a very broad-band seismometer on the surface of Mars. The Seismic Experiment for Interior Structure (SEIS) [2] has been collecting continuous data since early February 2019. The main focus of InSight is to enhance our understanding of the internal structure and dynamics of Mars, which includes the goal to better constrain the crustal thickness of the planet [3]. Knowing the present-day crustal thickness of Mars has important implications for its thermal evolution [4] as well as for the partitioning of silicates and heat-producing elements between the different layers of Mars. Current estimates for the crustal thickness of Mars are based on modeling the relationship between topography and gravity [5,6], but these studies rely on different assumptions, e.g. on the density of the crust and upper mantle, or the bulk silicate composition of the planet and the crust. The resulting values for the average crustal thickness differ by more than 100%, from 30 km to more than 100 km [7]. New independent constraints from InSight will be based on seismically determining the crustal thickness at the landing site. This single firm measurement of crustal thickness at one point on the planet will allow to constrain both the average crustal thickness of Mars as well as thickness variations across the planet when combined with constraints from gravity and topography [8]. Here we describe the determination of the crustal structure and thickness at the InSight landing site based on seismic receiver functions for three marsquakes compared with autocorrelations of InSight data [9].We acknowledge NASA, CNES, partner agencies and institutions (UKSA, SSO,DLR, JPL, IPGP-CNRS, ETHZ, IC, MPS-MPG) and the operators of JPL, SISMOC, MSDS, IRIS-DMC and PDS for providing SEED SEIS data. InSight data is archived in the PDS, and a full list of archives in the Geosciences, Atmospheres, and Imaging nodes is at https://pds-geosciences.wustl.edu/missions/insight/. This work was partially carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. ©2021, California Institute of Technology. Government sponsorship acknowledge

Seismic detection of the martian core

Clues to a planet's geologic history are contained in its interior structure, particularly its core. We detected reflections of seismic waves from the core-mantle boundary of Mars using InSight seismic data and inverted these together with geodetic data to constrain the radius of the liquid metal core to 1830 +/- 40 kilometers. The large core implies a martian mantle mineralogically similar to the terrestrial upper mantle and transition zone but differing from Earth by not having a bridgmanite-dominated lower mantle. We inferred a mean core density of 5.7 to 6.3 grams per cubic centimeter, which requires a substantial complement of light elements dissolved in the iron-nickel core. The seismic core shadow as seen from InSight's location covers half the surface of Mars, including the majority of potentially active regions-e.g., Tharsis-possibly limiting the number of detectable marsquakes.This is InSight contribution 200. We acknowledge NASA, CNES, and partner agencies and institutions (UKSA, SSO, ESA-PRODEX, DLR, JPL, IPGP-CNRS, ETHZ, IC, and MPS-MPG) for the development of SEIS. Numerical simulations were supported by a grant from the Swiss National Supercomputing Centre (CSCS) under project ID s922 as well as HPC resources of CINES under the allocation A0090407341, made by GENCI. We thank B. Dintrans, director of CINES, for his efficient handling of our request for computational time. Figures were created using matplotlib (83), seismic data processing was done in ObsPy (84), and numerical evaluation was done in NumPy and SciPy (85, 86). Funding: S.C.S., A.K., D.G., J.C., A.C.D., G.Z., and N.D. acknowledge support from ETHZ through the ETH+ funding scheme (ETH+2 19-1: “Planet MARS”). S.C.S. acknowledges funding from ETH research grant ETH-10 17-3. W.B.B., A.G.M., M.P.P., and S.E.S. were supported by the NASA InSight mission and funds from the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004). D.A. has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement 724690). The French teams acknowledge support from CNES as well as Agence Nationale de la Recherche (ANR-14-CE36-0012-02 and ANR-19-CE31-0008-08). A.R. was financially supported by the Belgian PRODEX program managed by the European Space Agency in collaboration with the Belgian Federal Science Policy Office. M.S. wishes to thank SANIMS (RTI2018-095594-B-I00). M.v.D. received support from the ERC under the European Union’s Horizon 2020 program (grant no. 714069). D.S. and C.S. acknowledge funding from ETH research grant ETH-06 17-02. J.C.E.I. acknowledges support from NASA grant 80NSSC18K1633. N.S., D.K., Q.H., R.M., V.L., and A.G.M. acknowledge NASA grant 80NSSC18K1628 for support. V.L. acknowledges support from the Packard Foundation. W.T.P. and C.C. received funding from the UK Space Agency, grant ST/S001239/1. A.H. was funded by the UK Space Agency (grant ST/R002096/1). A.-C.P. acknowledges the financial support and endorsement from the DLR Management Board Young Research Group Leader Program and the Executive Board Member for Space Research and Technology. Author contributions: S.C.S., D.G., S.C., R.F.G., Q.H., D.K., V.L., M.S., N.S., D.S., É.S., C.S., and G.Z. analyzed the seismic data and made ScS arrival time picks. S.C.S., P.L., D.G., Z.X., C.C., and W.T.P. performed the statistical analysis of the observed signals. S.C.S., Q.H., N.S., R.M., and A.G.M. identified the arrivals as ScS waves based on interior models from A.K., H.S., and A.R. A.K., M.D., A.C.D., and H.S. performed the inversions. S.C.S., A.K., P.L., D.G., D.A., J.C.E.I., M.K., C.P., A.-C.P., A.R., T.G., and S.E.S. participated and contributed to the interpretation of the results. Review of the continuous data and detection of marsquakes was done by S.C.S., S.C., G.Z., C.C., N.D., J.C., M.v.D., T.K., M.P., and A.H. with operational support by É.B., C.P., and P.M.D. S.C.S. and A.K. wrote the central part of the paper with contributions from H.S., N.S., D.A., J.C.E.I., A.G.M., A.-C.P., A.R., J.C., and M.v.D. J.C.E.I., R.M., M.K., and V.L. reviewed the contributions to the supplementary materials. The InSight mission is managed by W.B.B., M.P.P., and S.E.S. The SEIS instrument development was led by P.L., D.G., W.T.P., and W.B.B. Supplementary section 1 was written by M.S., D.S., and É.S. with contributions from S.C.S., C.S., and Z.X. Supplementary section 2 was written by D.K. and V.L. with contributions from J.C.E.I. and N.S. Supplementary section 3 was written by M.S. and É.S. Supplementary section 4 was written by R.F.G. with contributions from M.D. Supplementary section 5 was written by Q.H. with contributions from N.S. Supplementary section 6 was written by S.C.S. with contributions from the authors of the other supplements. Supplementary section 7 was written by Z.X. and C.C. with contributions from P.L. and W.T.P. Supplementary section 8 was written by A.K., M.D., A.C.D., and H.S. Supplementary section 9 was written by M.D. Supplementary section 10 was written by A.C.D., A.K., and M.D. Supplementary section 11 was written by D.A. and A.R. with contributions from A.K. Competing interests: The authors declare that they have no competing interests. Data and materials availability: We thank the operators of JPL, SISMOC, MSDS, IRIS-DMC, and PDS for providing SEED SEIS data (87). Three hundred interior models derived in this study are available from MSDS (88)

Largest recent impact craters on Mars: Orbital imaging and surface seismic co-investigation.

Two >130-meter-diameter impact craters formed on Mars during the later half of 2021. These are the two largest fresh impact craters discovered by the Mars Reconnaissance Orbiter since operations started 16 years ago. The impacts created two of the largest seismic events (magnitudes greater than 4) recorded by InSight during its 3-year mission. The combination of orbital imagery and seismic ground motion enables the investigation of subsurface and atmospheric energy partitioning of the impact process on a planet with a thin atmosphere and the first direct test of martian deep-interior seismic models with known event distances. The impact at 35°N excavated blocks of water ice, which is the lowest latitude at which ice has been directly observed on Mars

Surface waves and crustal structure on Mars

We detected surface waves from two meteorite impacts on Mars. By measuring group velocity dispersion along the impact-lander path, we obtained a direct constraint on crustal structure away from the InSight lander. The crust north of the equatorial dichotomy had a shear wave velocity of approximately 3.2 kilometers per second in the 5- to 30-kilometer depth range, with little depth variation. This implies a higher crustal density than inferred beneath the lander, suggesting either compositional differences or reduced porosity in the volcanic areas traversed by the surface waves. The lower velocities and the crustal layering observed beneath the landing site down to a 10-kilometer depth are not a global feature. Structural variations revealed by surface waves hold implications for models of the formation and thickness of the martian crust.D.K., S.C., D.G., J.C., C.D., A. K., S.C.S., N.D., and G.Z. were supported by the ETH+ funding scheme (ETH+02 19-1: “Planet Mars”). Marsquake Service operations at ETH Zürich were supported by ETH Research grant ETH-06 17-02. N.C.S. and V.L. were supported by NASA PSP grant no. 80NSSC18K1628. Q.H. and E.B. are funded by NASA grant 80NSSC18K1680. C.B. and J.L. were supported by NASA InSight PSP grant no. 80NSSC18K1679. S.D.K. was supported by NASA InSight PSP grant no. 80NSSC18K1623. P.L., E.B., M.D., H.S., E.S., M.W., Z.X., T.W., M.P., R.F.G. were supported by CNES and the Agence Nationale de la Recherche (ANR-19-CE31-0008-08 MAGIS) for SEIS operation and SEIS Science analysis. A.H., C.C. and W.T.P. were supported by the UKSA under grant nos. ST/R002096/1, ST/ W002523/1 and ST/V00638X/1. Numerical computations of McMC Approach 2 were performed on the S-CAPAD/DANTE platform (IPGP, France) and using the HPC resources of IDRIS under the allocation A0110413017 made by GENCI. A.H. was supported by the UKSA under grant nos. ST/R002096/1 and ST/W002523/1. F.N. was supported by InSight PSP 80NSSC18K1627. I.J.D. was supported by NASA InSight PSP grant no. 80NSSC20K0971. L.V.P. was funded by NASANNN12AA01C with subcontract JPL-1515835. The research was carried out in part by W.B.B., M.G. and M.P.P. at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004)Peer reviewe

The interior of Mars as seen by InSight (Invited)

InSight is the first planetary mission dedicated to exploring the whole interior of a planet using geophysical methods, specifically seismology and geodesy. To this end, we observed seismic waves of distant marsquakes and inverted for interior models using differential travel times of phases reflected at the surface (PP, SS...) or the core mantle-boundary (ScS), as well as those converted at crustal interfaces. Compared to previous orbital observations1-3, the seismic data added decisive new insights with consequences for the formation of Mars: The global average crustal thickness of 24-75 km is at the low end of pre-mission estimates5. Together with the the thick lithosphere of 450-600 km5, this requires an enrichment of heat-producing elements in the crust by a factor of 13-20, compared to the primitive mantle. The iron-rich liquid core is 1790-1870 km in radius6, which rules out the existence of an insulating bridgmanite-dominated lower mantle on Mars. The large, and therefore low-density core needs a high amount of light elements. Given the geochemical boundary conditions, Sulfur alone cannot explain the estimated density of ~6 g/cm3 and volatile elements, such as oxygen, carbon or hydrogen are needed in significant amounts. This observation is difficult to reconcile with classical models of late formation from the same material as Earth. We also give an overview of open questions after three years of InSight operation on the surface of Mars, such as the potential existence of an inner core or compositional layers above the CM