Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions

Abstract Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa–5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20–100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route

Denaturing mass photometry for straightforward optimization of protein-protein cross-linking reactions at single-molecule level

Mass photometry (MP) is a versatile, fast and low sample-consuming biophysical technique that gained interest in structural biology to study noncovalent assemblies in native conditions. We report here on a novel method to perform MP analysis in denaturing conditions (dMP) and its application for fast, accurate and straightforward optimization of chemical reactions in cross-linking mass spectrometry (XL-MS) workflows. dMP consists in a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. The proposed single-molecule method clearly overcomes the limitations and outperforms gold standard SDS-PAGE, as illustrated on several biological complexes. dMP provides an unprecedented and unmatched in-solution quantification of all coexisting XL species, including sub-complexes and non-specific XL aggregates, along with identification of significantly higher numbers of XL dipeptides in MS. We anticipate single-molecule dMP to be a high-impact game-changer for the XL-MS community with the potential to leverage the quality and reliability of XL-MS datasets

Severe acute respiratory infections (SARI) from influenza in adult patients in Chile: the experience of a sentinel hospital

Objective. To 1) describe clinical characteristics of adult patients in Chile with severe acute respiratory infections (SARI) associated with influenza viruses, and 2) analyze virus subtypes identified in specimens collected from those patients, hospital resources used in clinical management, clinical evolution, and risk factors associated with a fatal outcome, using observational data from the SARI surveillance network (SARInet). Methods. Adults hospitalized from 1 July 2011 to 31 December 2015 with influenza-associated SARI at a SARI sentinel surveillance hospital in Santiago were identified and the presence of influenza in all cases confirmed by reverse transcription polymerase chain reaction (RT-PCR), using respiratory samples. Results. A total of 221 patients (mean age: 74.1 years) were hospitalized with influenza-associated SARI during the study period. Of this study cohort, 91.4% had risk factors for complications and 34.3% had been vaccinated during the most recent campaign. Pneumonia was the most frequent clinical manifestation, occurring in 57.0% of the cohort; other manifestations included influenza-like illness, exacerbated chronic bronchitis, decompensated heart failure, and asthmatic crisis. Cases occurred year-round, with an epidemic peak during autumn–winter. Both influenza A (H1N1pdm09 and H3N2) and B virus co-circulated. Critical care beds were required for 26.7% of the cohort, and 19.5% needed ventilatory assistance. Multivariate analysis identified four significant factors associated with in-hospital mortality: 1) being bedridden (adjusted odds ratio (aOR): 22.3; 95% confidence interval (CI): 3.0–164); 2) admission to critical care unit (aOR: 8.9; CI: 1.44–55); 3) Pa02/Fi02 ratio 1 mg/dL) (aOR: 5.47; CI: 1.20–24). Seasonal influenza vaccine was identified as a significant protective factor (aOR: 0.14; CI: 0.021–0.90). Conclusions. Influenza-associated SARI affected mainly elderly patients with underlying conditions. Most patients evolved to respiratory failure and more than one-quarter required critical care beds. Clinical presentation was variable. Death was associated with host characteristics and disease-associated conditions, and vaccine was protective. Virus type did not influence outcome

Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner

The RPAP3 C-terminal domain identifies R2TP-like quaternary chaperones

International audienc

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis

International audienceThe PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly

The <i>A</i>. <i>baumannii</i> DSM30011 T6SS is required for out-competing <i>E</i>. <i>coli</i>.

<p>A) Representative image showing survival of DH5α <i>E</i>. <i>coli</i> after incubation in growth medium (control) or with <i>A</i>. <i>baumannii</i> ATCC17978 wild type and Δ<i>tssM</i> strains at a 1:1 ratio with or without the presence of a membrane (+M) and B) corresponding quantification (<i>N</i> = 6). C) Survival of DH5α <i>E</i>. <i>coli</i> after incubation in growth medium (control) or with <i>A</i>. <i>baumannii</i> DSM30011 wild type, Δ<i>tssM</i> and Δ<i>tssM</i> complemented strains at a 1:1 ratio, with D) corresponding quantification (<i>N</i> = 4). E) 18% SDS-PAGE (stained with Coomassie Blue) analysis of Hcp secretion in concentrated culture supernatants of <i>A</i>. <i>baumannii</i> DSM30011 wild type, Δ<i>tssM</i> and <i>tssM</i> complemented strains grown up to exponential phase in TSB. Molecular markers are indicated on the left. All quantifications are expressed as means ± SDM plotted in a logarithmic scale.</p

Competition between <i>A</i>. <i>baumannii</i> DSM30011 and nosocomial pathogens.

<p>Survival of A) different <i>P</i>. <i>aeruginosa</i> strains and C) rifampicin-resistant <i>K</i>. <i>pseumoniae</i> after incubation in growth medium (control) or with <i>A</i>. <i>baumannii</i> DSM30011 wild type, <i>tssM</i> deleted or complemented strains at a 10:1 ratio. <i>Pseudomonas</i> strains were selected with <i>Pseudomonas</i> Isolation Agar (PIA) media. Quantification is shown in B) and D), in which data are expressed as means ± SDM plotted in a logarithmic scale to visualize experimental variation from 3 independent experiments.</p

T6SS locus expression.

<p>A) RT-PCR transcriptional analysis of <i>hcp</i> and <i>tssM</i> expression in strains grown in L-broth; <i>rpoB</i> gene expression was used as endogenous control. The RNA not subjected to RT was also run in PCR (bottom panel, negative control) to ensure that PCR positive reactions were due to the presence of transcripts and not contaminating genomic DNA. B) The presence of Hcp (arrows) in concentrated culture supernatants of the indicated <i>A</i>. <i>baumannii</i> strains grown up to exponential phase in L-Broth was determined by 18% SDS-PAGE and Coomasie Blue staining. C) Control immunoblottings showing the presence of Hcp in the supernatant (first panel) and whole lysates fractions (second panel). A cell-lysis control using antibodies directed against EF-Tu (standard cytoplasmic marker) was performed using supernatant (third panel) or whole lysate (fourth panel) fractions. The final positions of the molecular mass markers (in kDa) are indicated on the right margin.</p