Biology subjects

Moreira, T.

Publications and source records attributed to Moreira, T..

6 recordsLinked to original sources

bioRxiv2026

The Holliday junction resolvase GEN1 preserves genome integrity and self-renewal in mouse embryonic stem cells

The maintenance of pluripotent stem cells (PSCs) under rapid proliferation requires mechanisms that both suppress replication-driven genome instability and preserve self-renewal capacity. Here, we show that, in contrast to somatic cells where it mainly acts as a backup, the Holliday junction resolvase GEN1 is required in mouse embryonic stem cells (ESCs), where its depletion severely compromises self-renewal and long-term maintenance. Loss of GEN1 induces the accumulation of cells with DNA content greater than 4C and chromosome fusions. Notably, a catalytically inactive GEN1 mutant rescues ESC colony formation, indicating that GEN1 supports ESC maintenance through non-enzymatic functions. In addition, GEN1 depletion increases ESC tolerance to topoisomerase I-mediated replication stress and renders this phenotype dependent on DNA-PK activity, suggesting that GEN1 loss alters how pluripotent cells cope with replication-associated DNA lesions. Together, these findings identify GEN1 as a non-redundant guardian of genome integrity in pluripotent cells, revealing both a catalysis-independent role in self-renewal and a contribution to the replication stress response, with implications for PSC genomic quality control. HighlightsO_LIIn contrast to somatic cells, GEN1 is specifically required for mouse pluripotent cell self-renewal and expansion in vitro. C_LIO_LIGEN1 loss induces accumulation of DNA content greater than 4C and chromosome fusions without loss of core pluripotency markers expression. C_LIO_LICatalytically inactive GEN1 mutant rescues ESC colony-forming capacity. C_LIO_LIGEN1 depletion increases ESC tolerance to topoisomerase I-mediated replication stress in a DNA-PK-dependent manner C_LI eTOCRamos-Lage et al. demonstrate that the resolvase GEN1 is essential for mouse embryonic stem cell self-renewal and genome stability. Strikingly, a catalytically dead mutant rescues colony formation, revealing an unexpected non-enzymatic role for GEN1 in pluripotency maintenance.

cell biology↗

bioRxiv2024

To 200,000 m/z and beyond: native electron capture charge reduction mass spectrometry deconvolves heterogeneous signals in large biopharmaceutical analytes

Great progress has been made in the detection of large biomolecular analytes by native mass spectrometry, however characterizing highly heterogeneous samples remains challenging due to the presence of many overlapping signals from complex ion distributions. Electron-capture charge reduction (ECCR), in which a protein cation captures free electrons without apparent dissociation, can dealign overlapping signals by shifting the ions to lower charge states. The concomitant shift to higher m/z also facilitates the exploration of instrument upper m/z limits if large complexes are used. Here we perform native ECCR on the bacterial chaperonin GroEL and megadalton scale adeno-associated virus (AAV) capsid assemblies on the Q Exactive UHMR mass spectrometer. Charge reduction of AAV8 capsids by up to 90% pushes signals well above 100,000 m/z and enables charge state resolution and mean mass determination of these highly heterogeneous samples, even for capsids loaded with genetic cargo. With minor instrument modifications the UHMR can detect charge reduced ion signals beyond 200,000 m/z. This work demonstrates the utility of ECCR for deconvolving heterogeneous signals in native mass spectrometry and presents the highest m/z signals ever recorded on an Orbitrap instrument, opening up the use of Orbitrap native mass spectrometry for heavier analytes than ever before.

molecular biology↗

bioRxiv2023

Regulation of inositol 5-phosphatase activity by the C2 domain of SHIP1 and SHIP2

SHIP1, an inositol 5-phosphatase, plays a central role in cellular signalling. As such, it has been implicated in many conditions. Exploiting SHIP1 as a drug target will require structural knowledge and the design of selective small molecules. We have determined apo, and magnesium and phosphate-bound structures of the phosphatase and C2 domains of SHIP1. The C2 domains of SHIP1 and the related SHIP2 modulate the activity of the phosphatase domain. To understand the mechanism, we performed activity assays, hydrogen-deuterium-exchange mass spectrometry and molecular dynamics on SHIP1 and SHIP2. Our findings demonstrate that the influence of the C2 domain is more pronounced for SHIP2 than SHIP1. We determined 91 structures of SHIP1 with fragments bound, with some near the interface between the two domains. We performed a mass spectrometry screen and determined four structures with covalent fragments. These structures could act as starting points for the development of potent, selective probes.

biochemistry↗

bioRxiv2020

The structural basis of fatty acid elongation by the ELOVL elongases

Very long chain fatty acids (VLCFAs) are essential building blocks for synthesis of the ceramides and sphingolipids required for nerve, skin and retina function and 3-keto acyl-CoA synthases (ELOVL elongases) perform the first step in the FA elongation cycle. Although ELOVLs are implicated in common diseases including insulin resistance, hepatic steatosis and Parkinsons, their underlying molecular mechanisms are unknown. Here we report the structure of the human ELOVL7 elongase, which includes an inverted transmembrane barrel structure surrounding a 35 [A] long tunnel containing a covalently-attached product analogue. The structure reveals the substrate binding sites in the tunnel and an active site deep in the membrane including the canonical ELOVL HxxHH sequence. This indicates a ping-pong mechanism for catalysis, involving unexpected covalent histidine adducts. The unusual substrate-binding arrangement and chemistry suggest mechanisms for selective ELOVL inhibition, relevant for diseases where VLCFAs accumulate such as X-linked adrenoleukodystrophy.

biochemistry↗

bioRxiv2020

Identification, Mapping and Relative Quantitation of SARS-Cov2 Spike Glycopeptides by Mass-Retention Time Fingerprinting

We describe a novel analytical method for rapid and robust identification, mapping and relative quantitation of glycopeptides from SARS-CoV-2 Spike protein. The method may be executed using any LC-TOF mass spectrometer, requires no specialised knowledge of glycan analysis and makes use of the differential resolving power of reversed phase HPLC. While this separation technique resolves peptides with high efficiency, glycans are resolved poorly, if at all. Consequently, glycopeptides consisting of the same peptide bearing different glycan structures will all possess very similar retention times and co-elute. While this has previously been viewed as a disadvantage, we show that shared retention time can be used to map multiple glycan species to the same peptide and location. In combination with MSMS and pseudo MS3, we have constructed a detailed mass-retention time database for Spike. This database allows any ESI-TOF equipped lab to reliably identify and quantify spike glycans from a single overnight elastase protein digest in less than 90 minutes.

biochemistry↗

bioRxiv2020

Targeting STAT3 signalling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer

PURPOSEEstrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Sulforaphane (SFN) has previously been shown to target CSCs but its mechanism of action is unclear. Here we investigate SFX-01, a stabilised formulation of SFN, for its effects on breast CSC activity in ER+ preclinical models and to study its mechanism. EXPERIMENTAL DESIGNCSC activity was measured by mammosphere formation efficiency (MFE), aldehyde dehydrogenase (ALDH) activity, and tumor formation using patient samples and patient-derived xenograft (PDX) tumors treated with SFX-01 alone or in combination with tamoxifen or fulvestrant. Gene expression and SFN target proteins in treated samples were assessed. RESULTSSFX-01 reduced MFE of both ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and ALDH activity of PDX tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor initiating cell frequency in secondary transplants at limiting dilution and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months endocrine treatment of ER+ patients (n=68) predicted poor prognosis. CONCLUSIONSOur data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

cancer biology↗