Identification of 113 new histone marks by CHiMA, a tailored database search strategy.

Shotgun proteomics has been widely used to identify histone marks. Conventional database search methods rely on the "target-decoy" strategy to calculate the false discovery rate (FDR) and distinguish true peptide-spectrum matches (PSMs) from false ones. This strategy has a caveat of inaccurate FDR caused by the small data size of histone marks. To address this challenge, we developed a tailored database search strategy, named "Comprehensive Histone Mark Analysis (CHiMA)." Instead of target-decoy-based FDR, this method uses "50% matched fragment ions" as the key criterion to identify high-confidence PSMs. CHiMA identified twice as many histone modification sites as the conventional method in benchmark datasets. Reanalysis of our previous proteomics data using CHiMA led to the identification of 113 new histone marks for four types of lysine acylations, almost doubling the number of previously reported marks. This tool not only offers a valuable approach for identifying histone modifications but also greatly expands the repertoire of histone marks.

A TRUSTED targeted mass spectrometry assay for pan-herpesvirus protein detection.

The presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. Here, we design and experimentally validate a mass spectrometry-based Targeted herpesviRUS proTEin Detection (TRUSTED) assay for monitoring human viruses representing the three Herpesviridae subfamilies-herpes simplex virus type 1, human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus. We demonstrate assay applicability for (1) capturing the temporal cascades of viral replication, (2) detecting proteins throughout a range of virus concentrations and in in vivo models of infection, (3) assessing the effects of clinical therapeutic agents and sirtuin-modulating compounds, (4) studies using different laboratory and clinical viral strains, and (5) discovering a role for carbamoyl phosphate synthetase 1 in supporting HCMV replication.

Temporal dynamics of protein complex formation and dissociation during human cytomegalovirus infection.

The co-evolution and co-existence of viral pathogens with their hosts for millions of years is reflected in dynamic virus-host protein-protein interactions (PPIs) that are intrinsic to the spread of infections. Here, we investigate the system-wide dynamics of protein complexes throughout infection with the herpesvirus, human cytomegalovirus (HCMV). Integrating thermal shift assays and mass spectrometry quantification with virology and microscopy, we monitor the temporal formation and dissociation of hundreds of functional protein complexes and the dynamics of host-host, virus-host, and virus-virus PPIs. We establish pro-viral roles for cellular protein complexes and translocating proteins. We show the HCMV receptor integrin beta 1 dissociates from extracellular matrix proteins, becoming internalized with CD63, which is necessary for virus production. Moreover, this approach facilitates characterization of essential viral proteins, such as pUL52. This study of temporal protein complex dynamics provides insights into mechanisms of HCMV infection and a resource for biological and therapeutic studies.

Tinagl1 Suppresses Triple-Negative Breast Cancer Progression and Metastasis by Simultaneously Inhibiting Integrin/FAK and EGFR Signaling.

Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.