Biology subjects

Becker, P.

Publications and source records attributed to Becker, P..

7 recordsLinked to original sources

bioRxiv2023

Mechanism of substrate binding and transport in BASS transporters

The Bile Acid Sodium Symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here we solve the crystal structure at 2.3 [A] of a transporter from Neisseria Meningitidis (ASBTNM) in complex with pantoate, a potential substrate of ASBTNM. The BASS family is characterised by two helices that cross-over in the centre of the protein in an arrangement that is intricately held together by two sodium ions. We observe that the pantoate binds, specifically, between the N-termini of two of the opposing helices in this cross-over region. During molecular dynamics simulations the pantoate remains in this position when sodium ions are present but is more mobile in their absence. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBTNM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism.

biochemistry↗

bioRxiv2023

Physical interaction between MSL2 and CLAMP assures direct cooperativity and prevents competition at composite binding sites

MSL2, the DNA-binding subunit of the Drosophila dosage compensation complex, cooperates with the ubiquitous protein CLAMP to bind MSL recognition elements (MREs) on the X chromosome. We explore the nature of the cooperative binding to these GA-rich, composite se-quence elements in reconstituted naive embryonic chromatin. We found that the cooperativity requires physical interaction between both proteins. Remarkably, disruption of this interaction does not lead to indirect, nucleosome-mediated cooperativity as expected, but to competition. The protein interaction apparently not only increases the affinity for composite binding sites, but also locks both proteins in a defined dimeric state that prevents competition. High Affinity Sites of MSL2 on the X chromosome contain variable numbers of MREs. We find that the cooperation between MSL2/CLAMP is not influenced by MRE clustering or arrangement, but happens largely at the level of individual MREs. The sites where MSL2/CLAMP bind strongly in vitro locate to all chromosomes and show little overlap to an expanded set of X-chromosomal MSL2 in vivo binding sites generated by CUT&RUN. Apparently, the intrinsic MSL2/CLAMP cooperativity is limited to a small selection of potential sites in vivo. This restriction must be due to components missing in our reconstitution, such as roX2 lncRNA.

molecular biology↗

bioRxiv2023

Dissecting the Determinants of Domain Insertion Tolerance and Allostery in Proteins

Domain insertion engineering is a promising approach to recombine the functions of evolutionarily unrelated proteins. Insertion of light-switchable receptor domains into a selected effector protein, for instance, can yield allosteric effectors with light-dependent activity. However, the parameters that determine domain insertion tolerance are poorly understood. Here, we used an unbiased screen to systematically assess the domain insertion permissibility of several evolutionary unrelated proteins. Training machine learning models on the resulting data allowed us to dissect features informative for domain insertion tolerance and revealed sequence conservation statistics as the strongest indicators of suitable insertion sites. Finally, extending our experimental pipeline towards the identification of switchable hybrids resulted in opto-chemogenetic derivatives of the transcription factor AraC that function as single-protein Boolean logic gates. Our study reveals determinants of domain insertion tolerance and facilitates the engineering of switchable proteins with unique mechanistic properties.

bioengineering↗

bioRxiv2022

The GEN-ERA toolbox: unified and reproducible workflows for research in microbial genomics .

BackgroundMicrobial culture collections play a key role in taxonomy by studying the diversity of their accessions and providing well characterized strains to the scientific community for fundamental and applied research. These microbial resource centers thus need to implement new standards in species delineation, including whole-genome sequencing and phylogenomics. In this context, the genomic needs of the Belgian Coordinated Collections of Microorganisms (BCCM) were studied, resulting in the GEN-ERA toolbox. The latter is a unified cluster of bioinformatic workflows dedicated to both bacteria and small eukaryotes (i.e. yeasts). FindingsThis public toolbox allows researchers without a specific training in bioinformatics to perform robust phylogenetic analyses. Hence, it facilitates all steps from genome downloading and quality assessment, including genomic contamination estimation, to tree reconstruction. It also offers workflows for average nucleotide identity comparisons and metabolic modeling. Technical detailsNextflow workflows are launched by a single command and are available on the GEN-ERA GitHub repository (https://github.com/Lcornet/GENERA). All the workflows are based on Singularity containers to increase reproducibility. TestingThe toolbox was developed for a diversity of microorganisms, including bacteria and fungi. It was further tested on an empirical dataset of 18 (meta)genomes of early-branching Cyanobacteria, providing the most up-to-date phylogenomic analysis of the Gloeobacterales order, the first group to diverge in the evolutionary tree of Cyanobacteria. ConclusionThe GEN-ERA toolbox can be used to infer completely reproducible comparative genomic and metabolic analyses on prokaryotes and small eukaryotes. Although designed for routine bioinformatics of culture collections, it can also be useful for other applications, as shown by our case study on Gloeobacterales.

bioinformatics↗

bioRxiv2022

Novel method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Long-read whole genome sequencing is prohibitively expensive for mtDNA heteroplasmy detection and often does not recapitulate the full mtDNA length. Here we describe a method to target, multiplex and sequence full-length, native single-molecule the human mitochondrial genome utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks as barcodes with long-read sequencing, we implemented a protocol in an optimal setting for both high or low integrity genomic DNA to target the circular mitochondrial genome with extremely high coverage. Our analytical pipeline efficiently detects single nucleotide heteroplasmy, physically determines phase and can accurately disentangle complex deletion patterns. This workflow is a unique tool for studying mtDNA variation in health and disease, and will accelerate mitochondrial research.

genomics↗

bioRxiv2021

Unsupervised discovery of dynamic cell phenotypic states from transmitted light movies

Identification of cell phenotypic states within heterogeneous populations, along with elucidation of their switching dynamics, is a central challenge in modern biology. Conventional single-cell analysis methods typically provide only indirect, static phenotypic readouts. Transmitted light images, on the other hand, provide direct morphological readouts and can be acquired over time to provide a rich data source for dynamic cell phenotypic state identification. Here, we describe an end-to-end deep learning platform, UPSIDE (for Unsupervised Phenotypic State IDEntification), for discovering cell states and their dynamics from transmitted light movies. UPSIDE uses the variational auto-encoder architecture to learn latent cell representations, that are then clustered for state identification, decoded for feature interpretation, and linked across movie frames for transition rate inference. Using UPSIDE, we identified distinct blood cell types in a heterogeneous dataset. From acute myeloid leukemia cell movies, we then identified stem-cell associated morphological states and their inter-conversion rates. UPSIDE opens up use of transmitted light movies for systematic exploration of cell state heterogeneity and dynamics in biology and medicine.

bioinformatics↗

bioRxiv2020

Augmented expansion of Treg cells from healthy and autoimmune subjects via adult progenitor cell co-culture.

Recent clinical experience has demonstrated that adoptive regulatory T cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPC) promote regulatory T cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a GMP compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is linked with a distinct Treg cell-intrinsic transcriptional program, characterized by diminished levels of core exhaustion (BATF, ID2, PRDM1, LAYN, DUSP1), and quiescence (TOB1, TSC22D3) related genes, coupled to elevated expression of cell-cycle and proliferation loci (MKI67, CDK1, AURKA, AURKB). In addition, MulTreg display a unique gut homing (CCR7lo {beta}7hi) phenotype and importantly, are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or Th1-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 TSDR demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno graft vs host disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

immunology↗