Björn Titz

Evolutionarily conserved herpesviral protein interaction networks.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposis sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

What do we learn from high-throughput protein interaction data?

The biological significance of protein interactions, their method of generation and reliability is briefly reviewed. Protein interaction networks adopt a scale-free topology that explains their error tolerance or vulnerability, depending on whether hubs or peripheral proteins are attacked. Networks also allow the prediction of protein function from their interaction partners and therefore, the formulation of analytical hypotheses. Comparative network analysis predicts interactions for distantly related species based on conserved interactions, even if sequences are only weakly conserved. Finally, the medical relevance of protein interaction analysis is discussed and the necessity for data integration is emphasized.

The protein network of bacterial motility

Motility is achieved in most bacterial species by the flagellar apparatus. It consists of dozens of different proteins with thousands of individual subunits. The published literature about bacterial chemotaxis and flagella documented 51 protein–protein interactions (PPIs) so far. We have screened whole genome two‐hybrid arrays of Treponema pallidum and Campylobacter jejuni for PPIs involving known flagellar proteins and recovered 176 and 140 high‐confidence interactions involving 110 and 133 proteins, respectively. To explore the biological relevance of these interactions, we tested an Escherichia coli gene deletion array for motility defects (using swarming assays) and found 159 gene deletion strains to have reduced or no motility. Comparing our interaction data with motility phenotypes from E. coli, Bacillus subtilis, and Helicobacter pylori, we found 23 hitherto uncharacterized proteins involved in motility. Integration of phylogenetic information with our interaction and phenotyping data reveals a conserved core of motility proteins, which appear to have recruited many additional species‐specific components over time. Our interaction data also predict 18 110 interactions for 64 flagellated bacteria.

The binary protein interactome of Treponema pallidum--the syphilis spirochete.

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed.

Transcriptional activators in yeast

Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested ∼6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix–loop–helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2).

MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells

Significance MITF, a master regulator of melanocytes and a major melanoma oncogene amplified in 30-40% of melanomas, determines proliferative or invasive phenotypes. Previously unrecognized as a driver of lysosomal biogenesis, we found that MITF expression correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis. This accumulation of incomplete organelles expands the late endosomal compartment, enhancing Wnt signaling by entrapping the Wnt machinery in multivesicular bodies. Wnt signaling can stabilize many proteins besides β-catenin. Our study identifies MITF as an oncogenic protein stabilized by Wnt, and describes three novel glycogen synthase kinase 3-regulated phosphorylation sites in this oncogene. This study deepens our knowledge on proliferative stages of melanoma: MITF, multivesicular bodies, and Wnt may form a feedback loop that drives proliferation. Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.

Metastatic castration-resistant prostate cancer reveals intrapatient similarity and interpatient heterogeneity of therapeutic kinase targets

Significance Metastatic castration-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. The need to identify new actionable targets in CRPC is crucial as we begin to examine the resistance mechanisms related to androgen withdrawal. Here, we report an unbiased quantitative phosphoproteomic approach to identify druggable kinases in metastatic CRPC. These kinase activation patterns revealed intrapatient similarity and interpatient heterogeneity across a large panel of targets. Interestingly, these kinase activities are not a result of mutation but rather pathway activation within the tumors themselves. The observation that similar kinase activities are present in most if not all anatomically disparate metastatic lesions from the same patient suggests that CRPC patients may benefit from individualized, targeted combination therapies. In prostate cancer, multiple metastases from the same patient share similar copy number, mutational status, erythroblast transformation specific (ETS) rearrangements, and methylation patterns supporting their clonal origins. Whether actionable targets such as tyrosine kinases are also similarly expressed and activated in anatomically distinct metastatic lesions of the same patient is not known. We evaluated active kinases using phosphotyrosine peptide enrichment and quantitative mass spectrometry to identify druggable targets in metastatic castration-resistant prostate cancer obtained at rapid autopsy. We identified distinct phosphopeptide patterns in metastatic tissues compared with treatment-naive primary prostate tissue and prostate cancer cell line-derived xenografts. Evaluation of metastatic castration-resistant prostate cancer samples for tyrosine phosphorylation and upstream kinase targets revealed SRC, epidermal growth factor receptor (EGFR), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), and MAPK1/3 and other activities while exhibiting intrapatient similarity and interpatient heterogeneity. Phosphoproteomic analyses and identification of kinase activation states in metastatic castration-resistant prostate cancer patients have allowed for the prioritization of kinases for further clinical evaluation.

Global Phosphoproteomics Reveals Crosstalk Between Bcr-Abl and Negative Feedback Mechanisms Controlling Src Signaling

Negative feedback fails to limit Src family kinase activity in the presence of Bcr-Abl, an oncoprotein that drives leukemia. When Negative Feedback Fails Bcr-Abl is a fusion protein with tyrosine kinase activity that causes some forms of leukemia. Bcr-Abl activates Src family tyrosine kinases (SFKs), but resistance to drugs, such as dasatinib and imatinib, that target Bcr-Abl and SFKs limits their clinical usefulness. With global tyrosine phosphoproteomic analysis in a murine leukemia cell line model, Rubbi et al. identified several negative feedback mechanisms that limited SFK activity and showed that their effectiveness was blunted by Bcr-Abl. Sensitivity of human leukemia cell lines to imatinib correlated with the amount of negative feedback signaling to SFKs. By exploring the mechanisms by which Bcr-Abl overpowered the negative feedback, the authors identified potential therapeutic targets for treating leukemias resistant to Bcr-Abl and SFK inhibitors or that may be combined with these tyrosine kinase inhibitors to prevent the development of resistance. In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, signaling by the Src family kinases (SFKs) critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling by quantitative mass spectrometry of Bcr-Abl–transformed cells in which the activities of the SFKs were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs and found that Bcr-Abl attenuated these inhibitory mechanisms. The C-terminal Src kinase (Csk)–binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl–mediated phosphorylation of the phosphatase Shp2 (Ptpn11), and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl–activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl–activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance.

BACH2 mediates negative selection and p53-dependent tumor suppression at the pre-B cell receptor checkpoint

The B cell–specific transcription factor BACH2 is required for affinity maturation of B cells. Here we show that Bach2-mediated activation of p53 is required for stringent elimination of pre-B cells that failed to productively rearrange immunoglobulin VH-DJH gene segments. After productive VH-DJH gene rearrangement, pre-B cell receptor signaling ends BACH2-mediated negative selection through B cell lymphoma 6 (BCL6)-mediated repression of p53. In patients with pre-B acute lymphoblastic leukemia, the BACH2-mediated checkpoint control is compromised by deletions, rare somatic mutations and loss of its upstream activator, PAX5. Low levels of BACH2 expression in these patients represent a strong independent predictor of poor clinical outcome. In this study, we demonstrate that Bach2+/+ pre-B cells resist leukemic transformation by Myc through Bach2-dependent upregulation of p53 and do not initiate fatal leukemia in transplant-recipient mice. Chromatin immunoprecipitation sequencing and gene expression analyses carried out by us revealed that BACH2 competes with BCL6 for promoter binding and reverses BCL6-mediated repression of p53 and other cell cycle checkpoint–control genes. These findings identify BACH2 as a crucial mediator of negative selection at the pre-B cell receptor checkpoint and a safeguard against leukemogenesis.

RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells.