In BriefPathway analysis of PTM data sets is typically performed at a gene-centric level because of the lack of appropriately curated PTM signature databases. We have developed a PTM signatures database (PTMsigDB) providing curated phosphorylation signatures of kinases, perturbations and signaling pathways to enable site-specific PTM signature enrichment analysis (PTM-SEA). Application of PTM-SEA to phosphoproteomes of several cell lines perturbed with growth factors, cell cycle inhibitors, or a specific PI3K inhibitor demonstrated the potential of our site centric approach to study dysregulated pathways in cancers. Graphical Abstract Highlights• Database of PTM site-specific phosphorylation signatures of kinases, perturbations and signaling pathways (PTMsigDB).• PTM signature enrichment analysis (PTM-SEA) outperformed gene-centric analysis in detection of EGF induced phospho signaling events.• PI3K perturbation signatures were readily detected in PI3Ka inhibited human breast cancer cells.• PTMsigDB and PTM-SEA can be freely accessed at https://github.com/broadinstitute/ssGSEA2.0.

HIV-1 infection of CD4 T cells leads to cytopathic effects and cell demise, which is counter to the observation that certain HIV-1-infected cells possess a remarkable long-term stability and can persist lifelong in infected individuals treated with suppressive antiretroviral therapy (ART). Using quantitative mass spectrometry-based proteomics, we showed that HIV-1 infection activated cellular survival programs that were governed by BIRC5, a molecular inhibitor of cell apoptosis that is frequently overexpressed in malignant cells. BIRC5 and its upstream regulator OX40 were upregulated in productively and latently infected CD4 T cells and were functionally involved in maintaining their viability. Moreover, OX40-expressing CD4 T cells from ART-treated patients were enriched for clonally expanded HIV-1 sequences, and pharmacological inhibition of BIRC5 resulted in a selective decrease of HIV-1-infected cells in vitro. Together, these findings suggest that BIRC5 supports long-term survival of HIV-1-infected cells and may lead to clinical strategies to reduce persisting viral reservoirs.

SignificanceThis work describes the most extensive discovery and functional characterization of small regulatory RNAs (sRNAs) in Mycobacterium tuberculosis to date. We comprehensively define the sRNAs expressed in M. tuberculosis under five host-like stress conditions. This reference dataset comprehensively defines the expression patterns and boundaries of mycobacterial sRNAs. We perform in-depth characterization of one sRNA, mycobacterial regulatory sRNA in iron (MrsI), which is induced in M. tuberculosis in multiple stress conditions. MrsI is critical for the iron-sparing response in mycobacteria by binding directly to mRNAs encoding nonessential iron-containing proteins to repress their expression. Interestingly, MrsI acts in an anticipatory manner, in which its induction by a variety of stresses primes M. tuberculosis to enter an iron-sparing state more rapidly upon iron deprivation.

Improved tuberculosis (TB) prevention and control depend critically on the development of a simple, readily accessible rapid triage test to stratify TB risk. We hypothesized that a blood protein-based host response signature for active TB (ATB) could distinguish it from other TB-like disease (OTD) in adult patients with persistent cough, thereby providing a foundation for a point-of-care (POC) triage test for ATB. Three adult cohorts consisting of ATB suspects were recruited. A bead-based immunoassay and machine learning algorithms identified a panel of four host blood proteins, interleukin-6 (IL-6), IL-8, IL-18, and vascular endothelial growth factor (VEGF), that distinguished ATB from OTD. An ultrasensitive POC-amenable single-molecule array (Simoa) panel was configured, and the ATB diagnostic algorithm underwent blind validation in an independent, multinational cohort in which ATB was distinguished from OTD with receiver operator characteristic–area under the curve (ROC-AUC) of 0.80 [95% confidence interval (CI), 0.75 to 0.85], 80% sensitivity (95% CI, 73 to 85%), and 65% specificity (95% CI, 57 to 71%). When host antibodies against TB antigen Ag85B were added to the panel, performance improved to 86% sensitivity and 69% specificity. A blood-based host response panel consisting of four proteins and antibodies to one TB antigen can help to differentiate ATB from other causes of persistent cough in patients with and without HIV infection from Africa, Asia, and South America. Performance characteristics approach World Health Organization (WHO) target product profile accuracy requirements and may provide the foundation for an urgently needed blood-based POC TB triage test.