Session TOF. There are 4 abstracts in this session.



Session: PROTEOMICS OF DISEASE: IMMUNITY AND INFECTIOUS DISEASE, time: 4:30 PM - 4:55 PM

Probes for Functional Proteomic Analysis of the Microbiome and Infectious Diseases

Matt Boygo

Hydrolases are enzymes (i.e. proteases, esterases, lipases) that often play important regulatory roles in many diverse types of infections by pathogens as well as in the regulation of commensal bacteria communities with the host. Therefore, tools that allow dynamic monitoring of their activity can be used as diagnostic agents, as imaging contrast agents and as proteomic tools for the identification of novel enzymes as drug leads. In this presentation, I will describe our efforts using small molecule activity-based probes (ABPs) to identify, inhibit and image various hydrolase targets in species of both commensal and pathogenic bacteria. We believe many of these enzymes are ideal targets to visualize and disrupt aspects of bacterial colonization and community formation inside a host.
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Session: PROTEOMICS OF DISEASE: IMMUNITY AND INFECTIOUS DISEASE, time: 4:55 PM - 5:20 PM

Discovering Tyrosine Phosphorylation in Mycobacterium Tuberculosis

Ulrike Kusebauch

Mycobacterium tuberculosis is the causative agent of tuberculosis, an infectious disease that remains a global health concern. While estimated one-quarter of the world's population has latent tuberculosis and is at risk of developing the disease, the human pathogen continues to claim ~1.5 million lives every year. Reversible protein phosphorylation is a well-known major regulatory mechanism across many life forms, including bacteria, which use this mechanism to sense and respond to changes in their environment. In M. tuberculosis, protein phosphorylation is the main signaling mechanism underlying the dynamic adaptive responses necessary for survival in its host. It is known that at least eleven two-component systems and eleven serine/threonine protein kinases mediate phosphorylation on aspartate, histidine, serine, and threonine. However, there was no conclusive evidence for protein phosphorylation on tyrosine, and tyrosine phosphorylation was thought to be absent in M. tuberculosis. I will discuss the discovery of a previously unrecognized phosphorylation system in M. tuberculosis using a combination of bacterial lysis, phospho-enrichment and highly sensitive mass spectrometry. We conclusively show extensive protein tyrosine phosphorylation of diverse M. tuberculosis proteins including serine/threonine protein kinases. Several serine/threonine protein kinases function as dual specificity kinases that phosphorylate tyrosine in cis and in trans, suggesting a major role of dual specificity kinases in bacterial phosphosignaling. Protein sequence point mutation of an activation segment phosphotyrosine site of the essential serine/threonine protein kinase PknB reduces PknB activity in vitro and in live Mtb, indicating a functional role for tyrosine phosphorylation in serine/threonine protein kinase regulation of bacterial growth. Together, our study provides the basis to understand how this new M. tuberculosis posttranslational modification affects physiology and pathogenesis.
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Session: PROTEOMICS OF DISEASE: IMMUNITY AND INFECTIOUS DISEASE, time: 5:20 PM - 5:35 PM

Recombinant MHC class I protein with isotope coded peptides enables relative and absolute quantification of the immunopeptidome

Lauren Stopfer1, 2; Josh Mesfin1; Forest White1
1Massachusetts Institute of Technology, Cambridge, MA; 2Koch Institute for Integrative Cancer Research, Cambridge, MA

Major histocompatibility (MHC) class I peptide antigens play a critical role in the recognition of malignant cells by the immune system.  Recent work has demonstrated that cancer cells modulate surface MHC levels in response to therapy, thereby affecting antitumor immunity. However, understanding the peptide repertoire response to treatment remains challenging, and is limited by quantitative strategies that lack the standard normalization controls used in other mass spectrometry-based methods. 

Here, we describe a novel quantitative immunopeptiomic approach that leverages recombinant heavy isotope coded peptide MHC complexes (hipMHCs) for relative and absolute quantitation of peptide repertoires using ultra low sample input (1x10^7cells). HipMHCs improve quantitative accuracy between analyses for both label free and multiplexed (TMT labeled) analyses by normalizing for variation and enabling regression against internal calibrants. Furthermore, for the first time we demonstrate absolute quantification using an internal standard curve for profiling copies per cell of any antigen of interest. 

We applied this platform to a biologically relevant context to interrogate how the immunopeptidome shifts with CDK4/6 inhibition, a known modulator of antigen presentation. We discovered that the intracellular response to CDK4/6i is directly reflected in the immunopeptidome, with peptides derived from proteins implicated in CDK4/6i response selectively positively and negatively enriched following stimulation. CDK4/6i mediated upregulation of MHC-I is thought to be modulated by the IFN-g response.  Intriguingly, IFN-g stimulation did not recapitulate the quantitative changes seen with CDK4/6i, but instead showed selective upregulation of IFN-g response peptides. 

Improved quanitative accuracy provided by hipMHCs reveal alterations in peptide repertoires that connect the intracellular response to extracellular immune presentation. Data generated by this method defines repertoire changes that in turn highlight targetable antigens of interest. We propose this platform can be leveraged to identify treatment modulated antigens for targeted immunotherapy, and inform combination therapy trial design.

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Session: PROTEOMICS OF DISEASE: IMMUNITY AND INFECTIOUS DISEASE, time: 5:35 PM - 5:50 PM

Temporal dynamics of protein complex formation and dissociation during viral infection

Xinlei Sheng; Yutaka Hashimoto; Laura Murray-Nerger; Ileana Cristea
Princeton University, Princeton, NJ

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. The spatial and temporal regulation of protein complexes and PPIs is fundamental to every step of an infection process, underlying both host defense and virus replication mechanisms. As a master manipulator of host cells, the ancient and large herpesvirus human cytomegalovirus (HCMV) induces a wide range of alterations to protein functions, accomplished primarily via finely-tuned protein interactions. Therefore, identifying these functional interactions and capturing their dynamics is critical for understanding viral infection and for developing antiviral therapies for this widely spread human pathogen. Here, we tackle the question of system-wide protein complex dynamics during the complete replication cycle of HCMV. To accomplish this, we use thermal proteome profiling (TPP) and thermal proximity coaggregation (TPCA) paired with multiplexed quantitative mass spectrometry, in conjunction with functional molecular virology and microscopy analyses. We monitor the temporal formation and dissociation of hundreds of functional protein complexes, as well as the dynamics of host-host, virus-host, and virus-virus PPIs during infection. We further overlay this dataset with our knowledge of protein subcellular localization and translocation during infection. Using this multi-disciplinary approach, we discover 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 facilitated characterization of essential viral proteins. For example, we uncover and validate the enhanced interactions of the viral packaging protein pUL52 with the interferon-inducible proteins IFIT1 and IFIT2 at the late stage of infection. Altogether, this global analysis of temporal protein complex dynamics provides insights into mechanisms of HCMV infection and a resource for biological and therapeutic studies.

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