Variability in protein cargo detection in technical and biological replicates of exosome-enriched extracellular vesicles

Exosomes are extracellular vesicles (EVs) of ~20–200 nm diameter that shuttle DNAs, RNAs, proteins and other biomolecules between cells. The large number of biomolecules present in exosomes demands the frequent use of high-throughput analysis. This, in turn, requires technical replicates (TRs), and biological replicates (BRs) to produce accurate results. As the number and abundance of identified biomolecules varies between replicates (Rs), establishing the replicate variability predicted for the event under study is essential in determining the number of Rs required. Although there have been few reports of replicate variability in high throughput biological data, none of them focused on exosomes. Herein, we determined the replicate variability in protein profiles found in exosomes released from 3 lung adenocarcinoma cell lines, H1993, A549 and H1975. Since exosome isolates are invariably contaminated by a small percentage of ~200–300 nm microvesicles, we refer to our samples as exosome-enriched EVs (EE-EVs). We generated BRs of EE-EVs from each cell line, and divided each group into 3 TRs. All Rs were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS) and customized bioinformatics and biostatistical workflows (raw data available via ProteomeXchange: PXD012798). We found that the variability among TRs as well as BRs, was largely qualitative (protein present or absent) and higher among BRs. By contrast, the quantitative (protein abundance) variability was low, save for the H1975 cell line where the quantitative variability was significant. Importantly, our replicate strategy identified 90% of the most abundant proteins, thereby establishing the utility of our approach

Variability in protein cargo detection in technical and biological replicates of exosome-enriched extracellular vesicles.

Exosomes are extracellular vesicles (EVs) of ~20-200 nm diameter that shuttle DNAs, RNAs, proteins and other biomolecules between cells. The large number of biomolecules present in exosomes demands the frequent use of high-throughput analysis. This, in turn, requires technical replicates (TRs), and biological replicates (BRs) to produce accurate results. As the number and abundance of identified biomolecules varies between replicates (Rs), establishing the replicate variability predicted for the event under study is essential in determining the number of Rs required. Although there have been few reports of replicate variability in high throughput biological data, none of them focused on exosomes. Herein, we determined the replicate variability in protein profiles found in exosomes released from 3 lung adenocarcinoma cell lines, H1993, A549 and H1975. Since exosome isolates are invariably contaminated by a small percentage of ~200-300 nm microvesicles, we refer to our samples as exosome-enriched EVs (EE-EVs). We generated BRs of EE-EVs from each cell line, and divided each group into 3 TRs. All Rs were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS) and customized bioinformatics and biostatistical workflows (raw data available via ProteomeXchange: PXD012798). We found that the variability among TRs as well as BRs, was largely qualitative (protein present or absent) and higher among BRs. By contrast, the quantitative (protein abundance) variability was low, save for the H1975 cell line where the quantitative variability was significant. Importantly, our replicate strategy identified 90% of the most abundant proteins, thereby establishing the utility of our approach

In Situ Humoral Immunity to Vimentin in HLA-DRB1*03+ Patients With Pulmonary Sarcoidosis

Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of HLA-DRB1*03, the Vα2.3/Vβ22 T-cell receptor (TCR), and Löfgren’s syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated in situ humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with HLA-DRB1*03. Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for HLA-DRB1*03 and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03+ patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4+ T-cells expressing the Vα2.3/Vβ22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (VimC-term) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03+ patient BALF contained higher concentrations of anti-VimC-term antibodies than BALF from both HCs and HLA-DRB1*03− patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked in situ recognition of vimentin by both T- and B-cells in HLA-DRB1*03+ sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus

presentation_1_In Situ Humoral Immunity to Vimentin in HLA-DRB1*03+ Patients With Pulmonary Sarcoidosis.PDF

<p>Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of HLA-DRB1*03, the Vα2.3/Vβ22 T-cell receptor (TCR), and Löfgren’s syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated in situ humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with HLA-DRB1*03. Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for HLA-DRB1*03 and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03⁺ patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4⁺ T-cells expressing the Vα2.3/Vβ22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (Vim_C-term) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03⁺ patient BALF contained higher concentrations of anti-Vim_C-term antibodies than BALF from both HCs and HLA-DRB1*03⁻ patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked in situ recognition of vimentin by both T- and B-cells in HLA-DRB1*03⁺ sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus.</p

Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets

Polyphosphate degradation by Nudt3-Zn 2+ mediates oxidative stress response

Polyphosphate (polyP) is a polymer of hundreds of phosphate residues present in all organisms. In mammals, polyP is involved in crucial physiological processes, including coagulation, inflammation, and stress response. However, after decades of research, the metabolic enzymes are still unknown. Here, we purify and identify Nudt3, a NUDIX family member, as the enzyme responsible for polyP phosphatase activity in mammalian cells. We show that Nudt3 shifts its substrate specificity depending on the cation; specifically, Nudt3 is active on polyP when Zn2+ is present. Nudt3 has in vivo polyP phosphatase activity in human cells, and importantly, we show that cells with altered polyP levels by modifying Nudt3 protein amount present reduced viability upon oxidative stress and increased DNA damage, suggesting that polyP and Nudt3 play a role in oxidative stress protection. Finally, we show that Nudt3 is involved in the early stages of embryo development in zebrafish.This work was supported by and is part of the I+D+i grant ref. PGC2018-096597-B-I00 (to J.J.) by the Spanish Ministerio de Ciencia e Innovación (MCIN). B.S.-M. was the recipient of a grant from the Agència de Gestió d’Ajuts Universitaris i de Recerca AGAUR ref. 2016FI_B 00025. H.J.J. was supported by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy (CIBBS, EXC-2189, Project ID 390939984)

Phosphoregulation of the oncogenic protein regulator of cytokinesis 1 (PRC1) by the atypical CDK16/CCNY complex.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that forms an active complex with cyclin Y (CCNY). Although both proteins have been recently implicated in cancer pathogenesis, it is still unclear how the CDK16/CCNY complex exerts its biological activity. To understand the CDK16/CCNY network, we used complementary proteomic approaches to identify potential substrates of this complex. We identified several candidates implicating the CDK16/CCNY complex in cytoskeletal dynamics, and we focused on the microtubule-associated protein regulator of cytokinesis (PRC1), an essential protein for cell division that organizes antiparallel microtubules and whose deregulation may drive genomic instability in cancer. Using analog-sensitive (AS) CDK16 generated by CRISPR-Cas9 mutagenesis in 293T cells, we found that specific inhibition of CDK16 induces PRC1 dephosphorylation at Thr481 and delocalization to the nucleus during interphase. The observation that CDK16 inhibition and PRC1 downregulation exhibit epistatic effects on cell viability confirms that these proteins can act through a single pathway. In conclusion, we identified PRC1 as the first substrate of the CDK16/CCNY complex and demonstrated that the proliferative function of CDK16 is mediated by PRC1 phosphorylation. As CDK16 is emerging as a critical node in cancer, our study reveals novel potential therapeutic targets