Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53.

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.

Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality.

Cathepsin B (CTSB) is a lysosomal endo- and exopeptidase that is also secreted in high amounts by malignant and non-malignant cells. We determined the effect of CTSB on the tumor cell secretome by shRNA-mediated silencing of CTSB mRNA expression and subsequent proteomic LC-MS/MS analysis of the cell supernatants. We identified significant protein changes of 17 secreted or shed proteins. Notably, we found a general reduction in protein abundance of ADAM10 substrates and lysosomal proteins. We corroborated reduced amounts of soluble ADAM10 (sADAM10) and soluble APP (sAPP) in the two cancer cell lines MDA-MB-231 and U2OS by immunoblotting. Interestingly, reductions in sADAM10 and sAPP could be reversed by re-introducing a catalytically inactive variant of CTSB, suggesting a formerly unknown non-catalytic function of the protease.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Community-Driven Data Analysis Training for Biology.

The primary problem with the explosion of biomedical datasets is not the data, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences. This project is accessible at https://training.galaxyproject.org.

[Dorsal ligamentoplasty for posttraumatic scapholunate dissociation]. / Die dorsale Bandplastik bei posttraumatischer skapholunärer Dissoziation.

Scapholunate dissociation in stages 2 and 3 may lead to posttraumatic damage if left untreated. Different types of treatment have been described, including capsulodesis, tenodesis, and others. The purpose of all procedures is to avoid palmar shift of the distal scaphoid resulting in secondary arthritis (SLAC wrist). This study aimed to evaluate a modified dorsal ligamentoplasty in second- and third-degree scapholunate dissociation in a prospective study design. Furthermore, the study aimed to find out if there is a correlation between radiological and clinical parameters at follow-up and whether or not a SLAC wrist can be avoided. From 2007 to 2011 we performed 29 dorsal ligamentoplasties in our hospital in patients with second- and third-degree scapholunate dissociation. In a prospective study design, 25 patients were followed up after an average period of 18 months. Parameters recorded preoperatively included ROM, the radiological parameters SL diastasis, SL diastasis in power grip and SL angle, the DASH score, and the VAS score. The postoperative evaluation additionally included power grip, the Mayo wrist score, and patients' subjective satisfaction. The average subjective satisfaction after surgery was good. The average postoperative ROM was 83.8 % of the ROM of the healthy wrist and decreased by 11 % compared with the preoperative level. The average postoperative grip strength was 85.7 % of the strength of the healthy hand. The VAS score was 2.7 with a significant reduction from 6.7 preoperatively. The average DASH score decreased significantly from 60 preoperatively to 22.5 postoperatively. The average Mayo wrist score after surgery was 73. All radiological parameters at follow-up were significantly better than preoperatively, although they remained in a pathological range. The Pearson correlation analysis showed no relevant correlation between clinical and radiological scores. Three cases (12 %) required revision surgery. The described modified dorsal ligamentoplasty at the carpus can be recommended in second- and third-degree scapholunate dissociation. Clinical results are comparable to other techniques, but there is only a single approach required and the complication rate is very low. The short average follow-up of 18 months must be viewed critically as the rate of SLAC wrists might be higher during longer follow-up periods.

Proteomic analysis of lung metastases in a murine breast cancer model reveals divergent influence of CTSB and CTSL overexpression.

Studies in the MMTV-PyMT (PyMT) breast cancer mouse model have shown a strong influence of the lysosomal cysteine cathepsins B or L on lung metastasis formation. Transgenic expression of human CTSB (tgCTSB) or CTSL (tgCTSL) both led to similar metastatic phenotypes with increased metastatic burden in the PyMT mice. However, recent studies in other tumor models proved marked differences in effects of either cathepsin on the proteome composition. We sought to analyze and compare proteome changes in the metastatic proteome of PyMT mice expressing either tgCTSB or tgCTSL to evaluate similarities and differences in those models. Performing an explorative, quantitative proteome comparison based on LC-MS/MS, we identified up to 3,000 proteins from murine lung metastases in three independent biological replicates per genotype. In both cases, when compared to wild-type (WT) mice, we noticed a pronounced impact of transgene cathepsin expression on the metastasis proteome. Highlights include increased moesin, integrin beta 1 and vinexin levels in the tgCTSB dataset and increased saposin and granulin levels in the tgCTSL dataset. Importantly, non-supervised hierarchical clustering clearly separated tgCTSB vs. tgCTSL induced proteome changes. In summary, tgCTSB and tgCTSL both display a strong and distinct impact on proteome composition of lung macrometastases in the PyMT model. Our observations suggest that they impact malignant behavior in distinct ways, thus further emphasizing interest into their tumor-contextual functionality.

Toward improved peptide feature detection in quantitative proteomics using stable isotope labeling.

Reliable detection of peptides in LC-MS data is a key algorithmic step in the analysis of quantitative proteomics experiments. While highly abundant peptides can be detected reliably by most modern software tools, there is much less agreement on medium and low-intensity peptides in a sample. The choice of software tools can have a big impact on the quantification of proteins, especially for proteins that appear in lower concentrations. However, in many experiments, it is precisely this region of less abundant but substantially regulated proteins that holds the biggest potential for discoveries. This is particularly true for discovery proteomics in the pharmacological sector with a specific interest in key regulatory proteins. In this viewpoint article, we discuss how the development of novel software algorithms allows us to study this region of the proteome with increased confidence. Reliable results are one of many aspects to be considered when deciding on a bioinformatics software platform. Deployment into existing IT infrastructures, compatibility with other software packages, scalability, automation, flexibility, and support need to be considered and are briefly addressed in this viewpoint article.

miR-200c dampens cancer cell migration via regulation of protein kinase A subunits.

Expression of miR-200c is a molecular switch to determine cellular fate towards a mesenchymal or epithelial phenotype. miR-200c suppresses the early steps of tumor progression by preventing epithelial-mesenchymal transition (EMT) and intravasation of tumor cells. Unraveling the underlying molecular mechanisms might pinpoint to novel therapeutic options. To better understand these mechanisms it is crucial to identify targets of miR-200c. Here, we employ a combination of quantitative proteomic and bioinformatic strategies to identify novel miR-200c targets. We identify and confirm two subunits of the central cellular kinase protein kinase A (PKA), namely PRKAR1A and PRKACB, to be directly regulated by miR-200c. Notably, siRNA-mediated downregulation of both proteins phenocopies the migratory behavior of breast cancer cells after miR-200c overexpression. Patient data from publicly accessible databases supports a miR-200c-PKA axis. Thus, our study identifies the PKA heteroprotein as an important mediator of miR-200c induced repression of migration in breast cancer cells. By bioinformatics, we define a miRNA target cluster consisting of PRKAR1A, PRKAR2B, PRKACB, and COF2, which is targeted by a group of 14 miRNAs.

Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits ß4 and ß1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the ß4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.