Biology subjects

Wan, M.

Publications and source records attributed to Wan, M..

4 recordsLinked to original sources

bioRxiv2019

Single-cell analyses identify tobacco smoke exposure-associated, dysfunctional CD16+ CD8 T cells with high cytolytic potential in peripheral blood

Tobacco smoke exposure has been found to impact immune response, leukocyte subtypes, DNA methylation, and gene expression in human whole blood. Analysis with single cell technologies will resolve smoking associated (sub)population compositions, gene expression differences, and identification of rare subtypes masked by bulk fraction data. To characterize smoking-related gene expression changes in primary immune cells, we performed single-cell RNA sequencing (scRNAseq) on >45,000 human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Major cell type population frequencies showed strong correlation between scRNAseq and mass cytometry. Transcriptomes revealed an altered subpopulation of Natural Killer (NK)-like T lymphocytes in smokers, which expressed elevated levels of FCGR3A (gene encoding CD16) compared to other CD8 T cell subpopulations. Relatively rare in nonsmokers (median: 1.8%), the transcriptionally unique subset of CD8 T cells comprised 7.3% of PBMCs in smokers. Mass cytometry confirmed a significant increase (p = 0.03) in the frequency of CD16+ CD8 T cells in smokers. The majority of CD16+ CD8 T cells were CD45RA positive, indicating an effector memory re-expressing CD45RA T cell (TEMRA) phenotype. We expect that cigarette smoke alters CD8 T cell composition by shifting CD8 T cells toward differentiated functional states. Pseudotemporal ordering of CD8 T cell clusters revealed that smokers cells were biased toward later pseudotimes, and characterization of established markers in CD8 T cell subsets indicates a higher frequency of terminally differentiated cells in smokers than in nonsmokers, which corresponded with a lower frequency in naive CD8 T cells. Consistent with an end-stage TEMRA phenotype, FCGR3A-expressing CD8 T cells were inferred as the most differentiated cluster by pseudotime analysis and expressed markers linked to senescence. Examination of differentially expressed genes in other PBMCs uncovered additional senescence-associated genes in CD4 T cells, NKT cells, NK cells, and monocytes. We also observed elevated Tregs, inducers of T cell senescence, in smokers. Taken together, our results suggest smoking-induced, senescence-associated immune cell dysregulation contributes to smoking-mediated pathologies.

immunology↗

bioRxiv2019

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containing Legionella effectors

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases (DUBs) in the canonical ubiquitination pathway, here we show that two Legionella effectors, named DupA (deubiquitinase for PR-ubiquitination) and DupB, reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub (PR-Ub) from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these two genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella containing vacuoles (LCV). Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.\n\nStatement of significanceUbiquitination is a vital posttranslational modification in eukaryotes. A variety of microbial pathogens exploit this pathway during their infection. Legionella pneumophila, the causative bacterial pathogen of Legionnaires disease, has been show to hijack host ubiquitination pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing a novel type of Ub-dependent posttranslational modification, namely PR-ubiquitination. Here we report two new players, DupA and DupB, involved in this unconventional pathway. We found that DupA and DupB function as PR-Ub specific DUBs and play a role in regulating the PR-ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-ubiquitination pathway, but may also facilitate the future identification of PR-ubiquitination pathways in eukaryotes.

biochemistry↗

bioRxiv2019

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

biochemistry↗

bioRxiv2019

Traces of past transposable element presence in Brassicaceae genome dark matter

AO_SCPLOWBSTRACTC_SCPLOWTransposable elements (TEs) are mobile, repetitive DNA sequences that make the largest contribution to genome bulk. They thus contribute to the so-called "dark matter of the genome", the part of the genome in which nothing is immediately recognizable as biologically functional. We developed a new method, based on k-mers, to identify degenerate TE sequences. With this new algorithm, we detect up to 10% of the A. thaliana genome as derived from as yet unidentified TEs, bringing the proportion of the genome known to be derived from TEs up to 50%. A significant proportion of these sequences overlapped conserved non-coding sequences identified in crucifers and rosids, and transcription factor binding sites. They are overrepresented in some gene regulation networks, such as the flowering gene network, suggesting a functional role for these sequences that have been conserved for more than 100 million years, since the spread of flowering plants in the Cretaceous.

genomics↗