␣-Synuclein is central in Parkinson's disease pathogenesis. Although initially ␣-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted ␣-synuclein in neuronal homeostasis, we have generated wild-type ␣-synuclein and ␤-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of ␣-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, ␣-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography-mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted ␣-synuclein causes cell death of recipient neuronal cells, which can be reversed after ␣-synuclein immunodepletion from the CM. High-and low-molecular-weight ␣-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced ␣-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that ␣-synuclein secretion serves to amplify and propagate Parkinson's diseaserelated pathology.

Naïve CD4 + T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4 + T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

Highlights d First deep proteogenomic landscape of non-smoking lung adenocarcinoma in East Asia d Identified age, sex-related endogenous, and environmental carcinogen mutagenic processes d Proteome-informed classification distinguished clinical features within early stages d Protein networks identified tumorigenesis hallmarks, biomarkers, and druggable targets

SummaryAssessing the impact of genomic alterations on protein networks is fundamental in identifying the mechanisms that shape cancer heterogeneity. We have used isobaric labeling to characterize the proteomic landscapes of 50 colorectal cancer cell lines and to decipher the functional consequences of somatic genomic variants. The robust quantification of over 9,000 proteins and 11,000 phosphopeptides on average enabled the de novo construction of a functional protein correlation network, which ultimately exposed the collateral effects of mutations on protein complexes. CRISPR-cas9 deletion of key chromatin modifiers confirmed that the consequences of genomic alterations can propagate through protein interactions in a transcript-independent manner. Lastly, we leveraged the quantified proteome to perform unsupervised classification of the cell lines and to build predictive models of drug response in colorectal cancer. Overall, we provide a deep integrative view of the functional network and the molecular structure underlying the heterogeneity of colorectal cancer cells.

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR-Cas9 knockouts (Δ) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage, and performed whole-genome sequencing of 173 subclones. Δ OGG1, Δ UNG, Δ EXO1, Δ RNF168, Δ MLH1, Δ MSH2, Δ MSH6, Δ PMS1, and Δ PMS2 produced marked mutational signatures indicative of being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-dG elimination is sequence-context-specific while uracil clearance is sequence-context-independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C>A transversions), differential misincorporation by replicative polymerases (T>C and C>T transitions), and we propose a ‘reverse template slippage’ model for T>A transversions. Δ MLH1, Δ MSH6, and Δ MSH2 signatures were similar to each other but distinct from Δ PMS2 . Finally, we developed a classifier, MMRDetect, where application to 7,695 WGS cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies.