Biology subjects

Le Bihan, T.

Publications and source records attributed to Le Bihan, T..

4 recordsLinked to original sources

bioRxiv2024

de Novo Sequencing of Antibodies for Identification of Neutralizing Antibodies in Human Plasma Post SARS-CoV-2 Vaccination

We present a method for sequencing polyclonal IgG enriched from human plasma, employing a combination of de novo sequencing, proteomics, bioinformatics, protein separation, sequencing, and peptide separations. Our study analyzes a single patients IgG antibody response triggered by the Moderna Spikevax mRNA COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generated 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo sequencing, exhibited similar or higher binding affinities than the original natural polyclonal antibody. Our neutralization tests revealed that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies while highlighting the effectiveness and potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Our proposed approach is an advancement in characterizing the IgG response by directly investigating the circulating pool of IgG without relying exclusively on the B-cell repertoire or population. This is crucial as the B-cell analysis may not accurately represent the circulating antibodies. Interestingly, a large proportion (80 to 90%) of the human antibody sequences generated against SARS-CoV-2 in the literature have been derived solely from B-cell analysis. Therefore, the ability to offer a different perspective is crucial in gaining a comprehensive understanding of the IgG response. Significance StatementWe investigate human IgG targeting the receptor binding domain using de novo proteomics. The peripheral B-cell repertoire may not adequately cover all the circulating IgG for human IgG sequencing. Our approach overcomes this limitation by using a de novo protein sequencing on top of standard proteomics. We obtained distinct de novo sequences, showcasing our methods potential. The recombinant proteins we generate possess traits comparable to or surpassing the naturally occurring polyclonal antibodies (pAbs). This study highlights similarities and differences between IgG populations in blood and circulating B-cells, which is crucial for future biologics development.

immunology↗

bioRxiv2019

Circadian protein regulation in the green lineage II. The clock gene circuit controls a phospho-dawn in Arabidopsis thaliana.

24-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time-series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the phospho-dawn. Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1-over-expressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1-over-expression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase / phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating post-translational mechanisms in the transmission of circadian timing information in plants.

systems biology↗

bioRxiv2019

Shotgun proteomics analysis of nanoparticle-synthesising Desulfovibrio alaskensis in response to platinum and palladium

Platinum and palladium are much sought-after metals of global critical importance in terms of abundance and availability. At the nano-scale these metals are of even higher value due to their catalytic abilities for industrial applications. Desulfovibrio alaskensis is able to capture ionic forms of both of these metals, reduce them, and synthesize elemental nanoparticles. Despite this ability very little is known about the biological pathways involved in the formation of these nanoparticles. Proteomic analysis of D. alaskensis in response to platinum and palladium has highlighted those proteins involved in both the reductive pathways and the wider stress-response system. A core set of 13 proteins was found in both treatments and consisted of proteins involved in metal transport and reduction. There were also 7 proteins specific to either platinum or palladium. Over-expression of one of these platinum-specific genes, a NiFe hydrogenase small subunit (Dde_2137), resulted in the formation of larger nanoparticles. This study improves our understanding of the pathways involved in the metal resistance mechanism of Desulfovibrio and informs how we can tailor the bacterium for nanoparticle production, enhancing its application as a bioremediation tool and as way to capture contaminant metals from the environment. ImportanceBacteria, in particularly D. alaskensis, represent a biological and greener way to capture high value metals such as platinum group metals from environmental and industrial waste streams. The recovery of these metals in nanoparticle forms adds extra value to this process as they can be used in a variety of different industrial applications as they have exceptional catalytic capabilities. D. alaskensis ability to do this, has been widely reported, though very little is understood about the underlying protein and genetic components. It is by understanding the biological basis of this capability that we can further improve and adapt this bacterium to be better at bioremediation and to control its ability to do so.

microbiology↗

bioRxiv2018

Circadian protein regulation in the green lineage I. A phospho-dawn anticipates light onset before proteins peak in daytime.

Diel regulation of protein levels and protein modification had been less studied than transcript rhythms. Here, we compare transcriptome data under light-dark cycles to partial proteome and phosphoproteome data, assayed using shotgun mass-spectrometry, from the alga Ostreococcus tauri, the smallest free-living eukaryote. 10% of quantified proteins but two-thirds of phosphoproteins were rhythmic. Mathematical modelling showed that light-stimulated protein synthesis can account for the observed clustering of protein peaks in the daytime. Prompted by night-peaking and apparently dark-stable proteins, we also tested cultures under prolonged darkness, where the proteome changed less than under the diel cycle. The dark-stable, prasinophyte-specific proteins were also reported to accumulate when O. tauri formed lipid droplets. In the phosphoproteome, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation suggests that a clock-regulated phospho-dawn prepares green cells for daytime functions. Acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase, implicating the clock-related, casein kinases 1 and 2 in phase-specific regulation, alternating with the CMGC protein kinase family. Understanding the dynamic phosphoprotein network should be facilitated by the minimal kinome and proteome of O. tauri. The data are available from ProteomeXchange, with identifiers PXD001734, PXD001735 and PXD002909. This submission updates a previous version, posted on bioRxiv on 4th April 2018, as https://www.biorxiv.org/content/10.1101/287862v1 HighlightThe phosphorylation of most protein sites was rhythmic under light-dark cycles, and suggested circadian control by particular kinases. Day-peaking, rhythmic proteins likely reflect light-stimulated protein synthesis in this microalga.

plant biology↗