An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.Apoptosis is a cell death program integral to various biological processes such as tissue homeostasis and development (1). The ability of cancer cells to evade apoptosis is considered a driving feature that imparts a selective cellular advantage allowing cells to persist inappropriately (2). A major component of apoptotic evasion in cancer arises from the misregulation of two enzyme classes, protein kinases and caspases. Kinases transfer the γ-phosphate from ATP to proteins to alter substrate function, and caspases act as executioners of the apoptotic program by facilitating the demolition of cellular constituents by cleaving key structural and enzymatic proteins (3, 4). Attenuation of caspase activity arising through kinase-mediated post-translational modifications or genetic mutations or deletions can contribute to malignant phenotypes by blocking apoptotic progression (5, 6).Interestingly, numerous examples have implicated cross-talk between caspases and kinases as a major apoptotic regulatory mechanism, and anecdotal examples have been identified in which phosphorylation at P4, P2, and P1′ (see Fig. 1A for cleavage site nomenclature) has been shown to block cleavage and affect cellular phenotypes (6–12). Accordingly, phosphorylation-dependent regulation of caspase-mediated cleavage has been hypothesized as a global regulator of apoptotic progression, especially in the context of cancer, where hyperactive, oncogenic kinases may act to increase phosphosite occupancy within caspase cleavage motifs (7). Indeed, we previously tested this hypothesis using predictive peptide match programs and identified CK2 phosphorylation sites on caspase-3 that regulated its activation by caspase-8 and -9 (13).Open in a separate windowFig. 1.Workflow for the global, unbiased analysis of the integration of phosphorylation and caspase-mediated degradation. A, illustration of the cleavage site nomenclature for proteases. Caspases cleave the scissile bond between a P1 aspartic acid and the P1′ residue. B, HeLa cell lysates were treated with or without λ phosphatase and subjected to caspase treatment followed by dephosphorylation of the sample previously left phosphorylated. Primary amines on protein N termini and lysine residues were dimethylated using heavy (+34, open circles) or light (+28, black circles) formaldehyde. Samples were pooled and trypsinized, which exposed an amine on the N terminus of the internal tryptic peptide. These peptides are captured through reaction with an ∼80-kDa aldehyde-substituted polymer. Importantly, native protein N termini and neo-N termini generated by caspase cleavage are resistant to reaction with the polymer because their reactive amines have been blocked by dimethylation. Enrichment of the N-terminome then occurs via negative selection when the reacted polymer is filtered away using a 10-kDa cut-off spin column. LC-MS/MS analysis of isotopically dimethylated peptides then allows comparative analysis between caspase degradomes of phosphorylated and dephosphorylated lysates. Caspase substrates will be inferred through identification of those peptides with a P1 aspartic acid. In the event that there is no difference in caspase substrate proteolysis between phosphorylated and dephosphorylated samples, a peptide ratio of ∼1:1 will be observed in MS1 [1]. Of interest are those peptide pairs that deviate from a 1:1 ratio [2].To build on our predictive strategy, we devised an unbiased, proteomic methodology to identify novel proteins for which phosphorylation regulates cleavage via caspases. We measured the caspase degradome in the context of a native phosphoproteome and compared it to the caspase degradome generated from lysates formerly dephosphorylated with λ bacteriophage phosphatase. To identify these events, we utilized the N-terminomic workflow TAILS¹ (terminal amino isotopic labeling of substrates) (14). Comparative analysis of the caspase degradomes from phosphorylated and dephosphorylated lysates revealed Yap1 and Golgin-160 as caspase substrates negatively regulated by phosphorylation.Surprisingly, we also identified a number of caspase substrates for which cleavage is promoted by phosphorylation, and during the course of our study, Dix et al. (15) demonstrated that phosphorylation at P3 can promote the cleavage of caspase peptide substrates. Our proteomic screen highlighted MST3 as a caspase substrate positively regulated by phosphorylation; however, in contrast to results obtained for MST3 protein in lysates, phosphorylation exerted a negative influence on the cleavage of an MST3 peptide, as was the case for other peptides modeled after Yap1 and Golgin-160. Collectively, these data suggest that although inhibitory effects of phosphorylation can arise through phosphorylation of residues proximal to the cleavage site, the positive effect of phosphorylation may stem from determinants other than those near the scissile bond. Subsequently, to test the effect of phosphorylation throughout the entirety of the caspase motif, we systematically walked phosphoserine through the length of model caspase-3, -7, and -8 substrate peptides and found that phosphorylation was generally inhibitory to caspase cleavage. Again, these observations suggest that positive effects of phosphorylation on the caspase cleavage of proteins observed in lysates likely arise through modulated ternary protein structure. Overall, our studies demonstrate that N-terminomics approaches can be tailored to identify novel, hierarchical events controlling the cleavage of caspase substrates.     

A Genetic Screen in Drosophila Reveals Novel Cytoprotective Functions of the Autophagy-Lysosome Pathway

The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61α), and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1). We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms.     

Hitchhiking and the Population Genetic Structure of Avian Influenza Virus

Previous studies have revealed a major difference in the phylogenetic structure, extent of genetic diversity, and selection pressure between the surface glycoproteins and internal gene segments of avian influenza viruses (AIV) sampled from wild birds. However, what evolutionary processes are responsible for these strikingly different evolutionary patterns is unclear. To address this issue, we estimated the rate of evolutionary change and time of origin of each segment of AIV sampled globally. Strikingly, the internal segments of the sampled AIV strains possess common ancestors that existed less than 200 years ago. Similarly recent times of origin were observed for each of the individual subtypes within the HA, NA, and NS gene segments. Such a shallow history of genetic diversity suggests an evolutionary model in which the genetic structure of AIV is shaped by a combination of occasional selective sweeps in the HA and NA (and possibly NS) segments, coupled with transient genetic linkage to the internal gene segments.     

Experimental Evolution Reveals a Genetic Basis for Membrane-Associated Virus Release

Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus remains poorly understood. Importantly, the genetic basis and evolvability of membrane-associated viral shedding have not been investigated. To address this, we performed a directed evolution experiment using coxsackievirus B3, a model enterovirus, in which we repeatedly selected the free-virion or the fast-sedimenting membrane-associated viral subpopulations. The virus responded to this selection regime by reproducibly fixing a series of mutations that altered the extent of membrane-associated viral shedding, as revealed by full-genome ultra-deep sequencing. Specifically, using site-directed mutagenesis, we showed that substitution N63H in the viral capsid protein VP3 reduced the ratio of membrane-associated to free viral particles by 2 orders of magnitude. These findings open new avenues for understanding the mechanisms and implications of membrane-associated viral transmission.     

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.     

Protective Role of Gamma Interferon during the Recall Response to Influenza Virus

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-γ). We assessed the contribution of IFN-γ to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-γ−/− mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-γ−/− mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-γ−/− mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-γ−/− mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-γ−/− mice was not dramatically affected, and the percentage of CD4⁺ and CD8⁺ T cells was similar to that of wild-type mice. The T cells from IFN-γ−/− mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-γ−/− mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-γ during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.     

Cleavage Activation of the Human-Adapted Influenza Virus Subtypes by Matriptase Reveals both Subtype and Strain Specificities

Cleavage activation of the hemagglutinin (HA) precursor is an essential step in the influenza virus replication cycle that is driven by host cell proteases. HA cleavage activation is required for virus-endosome membrane fusion and the subsequent release of the influenza virus genome into the cytoplasm. Previous studies have determined that HA cleavage is most likely driven by either membrane-bound or extracellular trypsin-like proteases that reside in the respiratory tract. However, there is still uncertainty regarding which proteases are critical for HA cleavage in vivo. Therefore, further investigation of HA cleavage activation is needed in order to gain insight into the critical proteases involved. Matriptase is a member of the type II transmembrane serine protease family that is highly expressed in a membrane-bound form throughout the respiratory tract. One feature of matriptase is that, once activated, the catalytic domain is secreted into the extracellular space and so serves as a functional extracellular protease. In this study, we have determined that the secreted, catalytic domain of matriptase has the ability to cleave and activate HA from the influenza virus H1 subtype but not the H2 and H3 subtypes. Furthermore, matriptase selectively cleaved the HA of particular strains within the H1 subtype, revealing both subtype and H1 strain specificity. Matriptase was also found to activate thrombolytic zymogens that have been shown to cleave and activate the influenza virus HA. Our data demonstrate that matriptase has the ability to cleave HA directly or indirectly by activating HA-cleaving zymogens.     

Simulation and Prediction of the Adaptive Immune Response to Influenza A Virus Infection

The cellular immune response to primary influenza virus infection is complex, involving multiple cell types and anatomical compartments, and is difficult to measure directly. Here we develop a two-compartment model that quantifies the interplay between viral replication and adaptive immunity. The fidelity of the model is demonstrated by accurately confirming the role of CD4 help for antibody persistence and the consequences of immune depletion experiments. The model predicts that drugs to limit viral infection and/or production must be administered within 2 days of infection, with a benefit of combination therapy when administered early, and cytotoxic CD8 T cells in the lung are as effective for viral clearance as neutralizing antibodies when present at the time of challenge. The model can be used to investigate explicit biological scenarios and generate experimentally testable hypotheses. For example, when the adaptive response depends on cellular immune cell priming, regulation of antigen presentation has greater influence on the kinetics of viral clearance than the efficiency of virus neutralization or cellular cytotoxicity. These findings suggest that the modulation of antigen presentation or the number of lung resident cytotoxic cells and the combination drug intervention are strategies to combat highly virulent influenza viruses. We further compared alternative model structures, for example, B-cell activation directly by the virus versus that through professional antigen-presenting cells or dendritic cell licensing of CD8 T cells.Understanding how the immune system combats influenza virus infection and how the virus can affect the immune system is crucial to predicting and designing prophylactic and therapeutic strategies against the infection (58). Antigenic shift and antigenic drift alter the degree to which preexisting immunity can control the virus. These factors also influence whether different arms of the adaptive immune system can cross-react against new strains of the virus. For example, shifts of the hemagglutinin (HA) and neuraminidase (NA) protein sequences limit the ability of antibodies to neutralize new variants of the virus and may make cross-reactive T-cell responses to conserved viral proteins more important. Other viral proteins, such as NS1, affect both the induction of type I interferon as well as the susceptibility of infected cells to interferon-mediated inhibition of viral gene expression (43). The efficiencies of viral replication and cell-to-cell viral spread are altered by mutations in the viral matrix and polymerase genes, while the survival of infected cells can be altered by the viral PB1-F2 protein. These attributes are influenced by mutations in the viral matrix (50, 51) and polymerase (30, 69) genes, while the survival of infected cells can be altered by the viral PB1-F2 protein (17). The multigenic aspect of influenza virus pathogenesis makes experimental prediction difficult and time-consuming. Computer simulation tools would be useful to independently dissect the potential contribution and relative importance of each factor or to investigate unexpected scenarios that are difficult to replicate experimentally.Mathematical models and computer simulations have been widely used to study viral dynamics and immune responses to viral infections, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency viruses (SIV), lymphocytic choriomeningitis virus (19, 55, 60, 61), and influenza A virus (3, 7, 8, 13, 34, 35, 52). More complex compartmental models of the immune system (4, 23) and models incorporating differential delay equations (21, 48, 68) have been used to better reflect the time that cells reside in a particular compartment or the duration of transit between compartments. In this study, we sought to develop a two-compartment mathematical model to assess the individual contributions of antigen presentation and activation of naïve T and B cells by antigen-presenting cells (APC), CD4 T-cell help, CD8 T-cell-mediated cytotoxicity, B cells, and antibody to control influenza A virus (IAV) infection and to explore the influence of anatomical location. We developed a model which represented published experimental findings on primary influenza virus infection. More importantly, the model was used to explore alternative structures for interactions between virus and immune cells, for example, comparing virus kinetics when antigen delivery and immune cell priming occurred through direct interaction of virus and immune cells or through a cellular intermediate. The model predicts that, under some circumstances, changes affecting antigen presentation more strongly impacted viral kinetics than other viral or immune factors (28, 73, 75, 78). This model highlights the importance of the assumptions used to synthesize a model and gaps in our understanding of the immune response regulating primary influenza virus infection. We discuss the implications of these findings for future influenza virus research and theories of influenza virus virulence based on influenza virus-immune system interactions.