Project description:The control of p53 protein stability is critical to its tumor suppressor functions. The CREB Binding Protein (CBP) transcriptional coactivator co-operates with MDM2 to maintain normally low physiologic p53 levels in cells via an exclusively cytoplasmic ‘E4’ polyubiquitination activity. Utilizing mass spectrometry to identify nuclear and cytoplasmic CBP interacting proteins that regulate compartmentalized CBP E4 activity, we identified Deleted in Breast Cancer 1 (DBC1) as a stoichiometric CBP-interacting protein that negatively regulates CBP–dependent p53 polyubiquitination, stabilizes p53, and augments p53-dependent apoptosis. TCGA analysis demonstrated that solid tumors often retain wild type p53 alleles in conjunction with DBC1 loss, supporting the hypothesis that DBC1 is selected for disruption during carcinogenesis as a surrogate for p53 functional loss. As DBC1 maintains p53 stability in the nucleus where p53 exerts its tumor suppressive transcriptional function, replacement of DBC1 functionality in DBC1-deleted tumors might also enhance p53 function and chemosensitivity for therapeutic benefit.
Project description:Pili on the surface of Sulfolobus islandicus are used for a host of functions, and serve as receptors for certain archaeal viruses. We find that these pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in SDS or 5M guanidinium-HCl. We have used cryo-EM to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the map with bioinformatics without prior knowledge of the pilin sequence, an approach that should prove useful when looking at assemblies where all of the components may not be known. The atomic structure of the archaeal pilus was unusual due to almost a third of the residues being either threonine or serine and many hydrophobic surface residues. While the map showed specific glycosylation of only three residues, mass per unit length measurements suggested extensive glycosylation. We show that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is quite similar to archaeal flagellin, establishing common evolutionary origins.
Project description:The proteomic approach aimed at the identification of interaction partners for the human dual phosphatase and tumor suppressor CDC14B by Co-inmunoprecipitation (Co-IP) experiments. In this approach, CDC14B interactomes from unsynchronized and G2/M enriched cells were compared semi-quantitatively based on spectral counting. This approach yielded the deubiquitylase USP9X as a G2/M-specific CDC14B interactor which has been confirmed by Western-blot (WB).
Project description:Shunt infections lead to grave neurologic morbidity for patients especially when there is a delay in diagnosis. C. acnes is the third most common cause of cerebrospinal fluid (CSF) shunt infection and is underdiagnosed due to the difficulty in culturing this fastidious, slow growing pathogen. Currently the gold standard for diagnosis of CSF shunt infections is microbiologic culture, however, in the case of C. acnes diagnostic cultures may be falsely negative. Therefore, new diagnostic methods are needed. To investigate potential CSF biomarkers of C. acnes CSF shunt infection we adapted a previously published rat model of CSF shunt infection to C. acnes. We found elevated levels of IL-1β, IL-6, CCL2 and IL-10 in the CSF and brain tissues of animals implanted with C. acnes infected catheters compared to sterile controls at day 1 post-infection. We found modest increases in neutrophils in the CSF and to a greater extend the brain tissue of animals with C. acnes infection which mirrors the clinical findings in C. acnes shunt infection. Mass spectrometry revealed that the CSF proteome is altered during C. acnes shunt infection and changes over the course of infection with an acute phase and pathogen neutralization response at day 1 post-infection to a more biosynthetic and metabolic response at day 28 post-infection relating to healing. Collectively, these results demonstrate that it is possible to distinguish C. acnes infection from sterile post-operative inflammation and CSF proteins could be useful in a diagnostic strategy for this pathogen that is difficult to diagnose.
Project description:Cerebrospinal fluid (CSF) shunt infection is a common and devastating complication of the treatment of hydrocephalus. Timely and accurate diagnosis is essential as these infections can lead to long term neurologic consequences like seizures, decreased IQ and impaired school performance. Currently the diagnosis of shunt infection relies on bacterial culture, however, culture is not always accurate especially as these infections are frequently caused by bacteria capable of forming biofilms like Staphylococcus epidermidis, Cutibacterium acnes, and Pseudomonas aeruginosa and may have very few planktonic bacteria in the CSF to be picked up on culture. Therefore, there is a critical need to identify a new rapid, and accurate method for diagnosis of CSF shunt infection with broad bacterial species coverage to improve the long-term outcomes of children suffering from these infections.
Project description:CUX1, a homeodomain-containing transcription factor, is recurrently deleted or mutated in multiple tumor types. In myeloid neoplasms, CUX1 deletion or mutation carries a poor prognosis. We have previously established that CUX1 functions as a tumor suppressor in hematopoietic cells across multiple organisms. Others, however, have described oncogenic functions of CUX1 in solid tumors, often attributed to truncated CUX1 isoforms, p75 and p110. Given the clinical relevance, it is imperative to clarify these discrepant activities. Herein, we sought to determine the CUX1 isoforms expressed in hematopoietic cells, and find that they express the full-length p200 isoform. Through the course of this analysis, we found no evidence of the p75 alternative transcript in any cell type examined. Using an array of orthogonal approaches, including biochemistry, proteomics, CRISPR/Cas9 genomic editing, and analysis of functional genomics datasets across a spectrum of normal and malignant tissue types, we found no data to support the existence of the CUX1 p75 isoform generated by an alternative transcriptional start site. Based on these results, prior studies of p75 require reevaluation, including the interpretation of oncogenic roles attributed to CUX1.
Project description:Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.
Project description:PI3K is a heterodimer of p110 catalytic and p85 adaptor subunits that is activated by agonist-stimulated receptor tyrosine kinases. Although p85 recruits p110 to activated receptors on membranes, p85 loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. p110 localizes to microtubules via MAP4, facilitating its interaction with activated receptor kinases on endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85 knock down, the residual p110 coupled predominantly to p85 exhibits enhanced recruitment with receptor tyrosine kinases to endosomes. Moreover, the p110 C2 domain binds PI3P and this interaction is also required to recruit p110 to endosomes and for PI3K/Akt signaling. Stable knockdown of p85, which mimics the reduced p85levels observed in cancer, enhances cell growth and tumorsphere formation, and these effects are abrogated by MAP4 or p85knockdown, underscoring their role in the tumor-promoting activity of p85loss.
Project description:The dataset contains the first-ever comprehensive biocodicological analysis of medieval library books and chapters using zooarchaeological mass spectrometry (ZooMS). Here, we analyze 68 codices and 59 charters (1490 samples in total) from one single monastic institution, namely the Cistercian abbey of Orval in present-day Belgium. This dataset contains LC-MSMS analysis of samples from all charters (59 samples) and manuscript 22 (27 samples)