C7orf59/LAMTOR4 phosphorylation and structural flexibility modulate ragulator assembly

Ragulator is a pentamer composed of p18, MP1, p14, C7orf59, and hepatitis B virus X-interacting protein (HBXIP; LAMTOR 1-5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling. Here, we present the crystal structure of human HBXIP-C7orf59 dimer (LAMTOR 4/5) at 2.9 angstrom and identify a phosphorylation site on C7orf59 which modulates its interaction with p18. Additionally, we demonstrate the requirement of HBXIP-C7orf59 to stabilize p18 and allow further binding of MP1-p14. The structure of the dimer revealed an unfolded N terminus in C7orf59 (residues 1-15) which was shown to be essential for p18 binding. Full-length p18 does not interact stably with MP1-p14 in the absence of HBXIP-C7orf59, but deletion of p18 residues 108-161 rescues MP1-p14 binding. C7orf59 was phosphorylated by protein kinase A (PKA) in vitro and mutation of the conserved Ser67 residue to aspartate prevented phosphorylation and negatively affected the C7orf59 interaction with p18 both in cell culture and in vitro. C7orf59 Ser67 was phosphorylated in human embryonic kidney 293T cells. PKA activation with forskolin induced dissociation of p18 from C7orf59, which was prevented by the PKA inhibitor H-89. Our results highlight the essential role of HBXIP-C7orf59 dimer as a nucleator of pentameric Ragulator and support a sequential model of Ragulator assembly in which HBXIP-C7orf59 binds and stabilizes p18 which allows subsequent binding of MP1-p149915891602CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2014/12445-0; 2017/21455-7; 2014/17264-3190174/2012-

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

Identification and characterization of an alternatively spliced isoform of the human protein phosphatase 2Aα catalytic subunit

Submitted by Manoel Barata ([email protected]) on 2018-09-14T12:28:06Z No. of bitstreams: 1 JournalBiolChemistryzb4853.pdf: 1990964 bytes, checksum: bb68ee6b122af42a80fca5054d5c4a3d (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2018-09-14T19:47:44Z (GMT) No. of bitstreams: 1 JournalBiolChemistryzb4853.pdf: 1990964 bytes, checksum: bb68ee6b122af42a80fca5054d5c4a3d (MD5)Made available in DSpace on 2018-09-14T19:47:44Z (GMT). No. of bitstreams: 1 JournalBiolChemistryzb4853.pdf: 1990964 bytes, checksum: bb68ee6b122af42a80fca5054d5c4a3d (MD5) Previous issue date: 2012Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. / Universidade Estadual de Campinas. Instituto de Biologia. Campinas, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil.Universidade Estadual de Campinas. Centro de Biologia Molecular e Engenharia Genética, Campinas, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. / Universidade Estadual de Campinas. Instituto de Biologia. Campinas, SP, Brasil.Universidade Estadual de Campinas. Centro de Biologia Molecular e Engenharia Genética, Campinas, SP, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation

Crystal Structure of the human TIP41 Orthologue, TIPRL, reveals a novel fold and a binding site for the PP2AC C-TERMINUS

Submitted by Luciane Willcox ([email protected]) on 2017-04-11T19:37:48Z No. of bitstreams: 1 Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.pdf: 2132858 bytes, checksum: f3c011ca732ddc9cb6872d8a9f79a48c (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2017-07-26T14:29:12Z (GMT) No. of bitstreams: 1 Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.pdf: 2132858 bytes, checksum: f3c011ca732ddc9cb6872d8a9f79a48c (MD5)Made available in DSpace on 2017-07-26T14:29:12Z (GMT). No. of bitstreams: 1 Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.pdf: 2132858 bytes, checksum: f3c011ca732ddc9cb6872d8a9f79a48c (MD5) Previous issue date: 2016Institute of Chemistry, University of Campinas, Campinas, SP, Brazil. / Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil. / Institute of Biology, University of Campinas, Campinas, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil. / Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil.Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Diamond Light Source, Chilton, UK.Diamond Light Source, Chilton, UK.Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.Brazilian National Laboratory for Biosciences, Center for Research in Energy and Materials, Campinas, SP, Brazil.Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition