Heterodimers of functionally divergent ARF-GEF paralogues prevented by self-interacting dimerisation domain

Functionally divergent paralogs of homomeric proteins do not form potentially deleterious heteromers, which requires distinction between self and non-self (Hochberg et al., 2018; Marchant et al, 2019; Marsh and Teichmann, 2015). In Arabidopsis, two ARF guanine-nucleotide exchange factors (ARF-GEFs) related to mammalian GBF1, named GNOM and GNL1, can mediate coatomer complex (COPI)-coated vesicle formation in retrograde Golgi-endoplasmic reticulum (ER) traffic (Geldner et al., 2003; Richter et al., 2007; Teh and Moore, 2007). Unlike GNL1, however, GNOM is also required for polar recycling of endocytosed auxin efflux regulator PIN1 from endosomes to the plasma membrane. Here we show that these paralogues form homodimers constitutively but no heterodimers. We also address why and how GNOM and GNL1 might be kept separate. These paralogues share a common domain organisation and each N-terminal dimerisation (DCB) domain can interact with the complementary fragment (DDCB) of its own and the other protein. However, unlike self-interacting DCBGNOM (Grebe et al., 2000; Anders et al., 2008), DCBGNL1 did not interact with itself nor DCBGNOM. DCBGNOM removal or replacement with DCBGNL1, but not disruption of cysteine bridges that stabilise DCB-DCB interaction, resulted in GNOM-GNL1 heterodimers which impaired developmental processes such as lateral root formation. We propose precocious self-interaction of the DCBGNOM domain as a mechanism to preclude formation of fitness-reducing GNOM-GNL1 heterodimers.

Deformation Parameters and Collective Temperature Changes in Photofission Mass Yields of Actinides Within the Systematic Statistical Scission Point Model

The photofission fragment mass yields of actinides are evaluated using a systematic statistical scission point model. In this model, all energies at the scission point are presented as a linear function of the mass numbers of fission fragments. The mass yields are calculated with a new approximated relative probability for each complementary fragment. The agreement with the experimental data is quite good, especially with a collective temperature Tcol of 2 MeV at intermediate excitation energy and Tcol = 1 MeV for spontaneous fission. This indicates that the collective temperature is greater than the value obtained by the initial excitation energy. The generalized superfluid model is applied for calculating the fragment temperature. The deformation parameters of fission fragments have been obtained by fitting the calculated results with the experimental values. This indicates that the deformation parameters decrease with increasing excitation energy. Also, these parameters decrease for fissioning systems with odd mass numbers.

Proteomic Database Search Engine for Two-Dimensional Partial Covariance Mass Spectrometry

<div>We present a protein database search engine for the automatic identi?cation of peptide and protein sequences using the recently introduced method of two-dimensional partial covariance mass spectrometry (2D-PC-MS). Since 2D-PC-MS measurement reveals correlations between fragments stemming from the same or consecutive decomposition processes, the ?first-of-its-kind 2D-PC-MS search engine is based entirely on the direct matching of the pairs of theoretical and the experimentally detected correlating fragments, rather than of individual fragment signals or their series. We demonstrate that the high structural speci?city a?orded by 2D-PC-MS fragment correlations enables our search engine to reliably identify the correct peptide sequence, even from a spectrum with a large proportion of contaminant signals. While for peptides the 2D-PC-MS correlation matching procedure is based on complementary and internal ion correlations, the identi?cation of intact proteins is entirely based on the ability of 2D-PC-MS to spatially separate and resolve the experimental correlations between complementary fragment ions.</div>

Proteomic Database Search Engine for Two-Dimensional Partial Covariance Mass Spectrometry

<div>We present a protein database search engine for the automatic identi?cation of peptide and protein sequences using the recently introduced method of two-dimensional partial covariance mass spectrometry (2D-PC-MS). Since 2D-PC-MS measurement reveals correlations between fragments stemming from the same or consecutive decomposition processes, the ?first-of-its-kind 2D-PC-MS search engine is based entirely on the direct matching of the pairs of theoretical and the experimentally detected correlating fragments, rather than of individual fragment signals or their series. We demonstrate that the high structural speci?city a?orded by 2D-PC-MS fragment correlations enables our search engine to reliably identify the correct peptide sequence, even from a spectrum with a large proportion of contaminant signals. While for peptides the 2D-PC-MS correlation matching procedure is based on complementary and internal ion correlations, the identi?cation of intact proteins is entirely based on the ability of 2D-PC-MS to spatially separate and resolve the experimental correlations between complementary fragment ions.</div>

A Mass Spectrometry-Cleavable N-Terminal Tagging Strategy for System-Level Protease Activity Profiling

Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. This irreversible PTM is regulated and catalyzed by proteases through hydrolysis reaction in the process of protein maturation or degradation. The identification of proteolytic substrates is pivotal in understanding the specificity of proteases and the physiological role of proteolytic process. However, tracking these biological phenomena in native cells is very difficult due to their low abundances. Currently, no efficient methods are available to identify proteolytic products from large-scale samples. To facilitate the targeted identification of low-abundant proteolytic products, we devise a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-terminus. Within the PFP-Rink-biotin reagent, an MS-cleavable feature is designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. This is the first reported study of identifying proteolytic N-terminus by MS-cleavable feature widely adopted in studying low-abundant protein PTMs and cross-linking/MS. The proof-of-concept study was performed with multiple standard proteins whose N-terminus were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia.coli cells were demonstrated along with a bioinformatics workflow. We believe this method can be very useful to locate proteolytic products in native cellular environment with high confidence.<br>

A Mass Spectrometry-Cleavable N-Terminal Tagging Strategy for System-Level Protease Activity Profiling

Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. This irreversible PTM is regulated and catalyzed by proteases through hydrolysis reaction in the process of protein maturation or degradation. The identification of proteolytic substrates is pivotal in understanding the specificity of proteases and the physiological role of proteolytic process. However, tracking these biological phenomena in native cells is very difficult due to their low abundances. Currently, no efficient methods are available to identify proteolytic products from large-scale samples. To facilitate the targeted identification of low-abundant proteolytic products, we devise a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-terminus. Within the PFP-Rink-biotin reagent, an MS-cleavable feature is designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. This is the first reported study of identifying proteolytic N-terminus by MS-cleavable feature widely adopted in studying low-abundant protein PTMs and cross-linking/MS. The proof-of-concept study was performed with multiple standard proteins whose N-terminus were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia.coli cells were demonstrated along with a bioinformatics workflow. We believe this method can be very useful to locate proteolytic products in native cellular environment with high confidence.<br>

Recent developments of the FALSTAFF experimental setup

The study of nuclear fission is encountering renewed interest with the development of GEN-IV reactor concepts, mostly working in the neutron fast energy domain. To support the fast reactor technologies, new high quality nuclear data are needed. New facilities are being constructed to produce high intensity neutron beams from hundreds of keV to few tens of MeV (Licorne, NFS, nELBE, ...). They will open new opportunities to provide nuclear data. In this framework the development of an experimental setup called FALSTAFF for a characterisation of actinide fission fragments has been undertaken. Fission fragment yields and associated neutron multiplicities will be measured as a function of the neutron energy. Based on time-of-flight and residual energy technique, the setup will allow the simultaneous measurement of the complementary fragment velocity and energy. The FALSTAFF setup and the upgrade of the first arm prototype with the new ionisation chamber CALIBER will be presented. The performances of the experimental apparatus is discussed.

Molecular Species Delimitation of Icelandic Brittle Stars (Ophiuroidea)

Brittle stars (Echinodermata: Ophiuroidea) comprise over 2, 000 species, all of which inhabit marine environments and can be abundant in the deep sea. Morphological plasticity in number and shape of skeletal parts, as well as variable colors, can complicate correct species identification. Consequently, DNA sequence analysis can play an important role in species identification. In this study we compared the genetic variability of the mitochondrial cytochrome c subunit I gene (COI) and the nuclear small subunit ribosomal DNA (SSU, 18S rDNA) to morphological identification of 66 specimens of 11 species collected from the North Atlantic in Icelandic waters. Also two species delimitation tools, Automatic Barcode Gap Discovery (ABGD) and General Mixed Yule Coalescence Method (GMYC) were performed to test species hypotheses. The analysis of both gene fragments was successful to discriminate between species and provided new insights into some morphological species hypothesis. Although less divergent than COI, it is helpful to use the SSU region as a complementary fragment to the barcoding gene.

Fragment Screening by Native State Mass Spectrometry

Native state mass spectrometry (MS) has been recognised as a rapid, sensitive, and high throughput method to directly investigate protein-ligand interactions for some time, however there are few examples reporting this approach as a screening method to identify relevant protein–fragment interactions in fragment-based drug discovery (FBDD). In this paper an overview of native state MS will be presented, highlighting the attractive properties of this method within the context of fragment screening applications. A summary of published examples using MS for fragment screening will be described and reflection on the outlook for the future adoption and implementation of native state MS as a complementary fragment screening method will be presented.