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Specific activating missense HRAS variants cause Costello syndrome (CS), a RASopathy with recognizable facial features. The majority of these dominant disease causing variants affect the glycine residues in position 12 or 13. A clinically suspected CS diagnosis can be confirmed through identification of a dominant pathogenic HRAS variant. A novel HRAS variant predicting p.(Glu62_Arg68dup) was identified in an individual with hypertrophic cardiomyopathy, Chiari 1 malformation and ectodermal findings consistent with a RASopathy. Functional studies showed that the p.Glu62_Arg68dup alteration affects HRAS interaction with effector protein PIK3CA (catalytic subunit of phosphoinositide 3-kinase) and the regulator neurofibromin 1 (NF1) GTPase-activating protein (GAP). HRAS(Glu62_Arg68dup) binding with effectors rapidly accelerated fibrosarcoma (RAF1), RAL guanine nucleotide dissociation stimulator (RALGDS) and phospholipase C1 (PLCE1) was enhanced. Accordingly, p.Glu62_Arg68dup increased steady-state phosphorylation of MEK1/2 and ERK1/2 downstream of RAF1, whereas AKT phosphorylation downstream of PI3K was not significantly affected. Growth factor stimulation revealed that expression of HRAS(Glu62_Arg68dup) abolished the HRAS’ capacity to modulate downstream signaling. Our data underscore that different qualities of dysregulated HRAS-dependent signaling dynamics determine the clinical severity in CS.

The muscle-associated respiratory protein myoglobin (MB) is expressed in multiple types of cancer, including breast and prostate tumors. In Kaplan-Meier analyses of the two tumor types, MB positivity is associated with favorable prognoses. Despite its well-characterized function in myocytes, the role of MB in cancer remains unclear. To study the impact of endogenous MB expression, small interfering RNA MB-knockdown cells were engineered using breast, prostate and colon cancer cell lines (MDA-MB468, LNCaP, DLD-1), and their transcriptomes were investigated using RNA-Seq at different oxygen levels. In MB-positive cells, increased expression of glycolytic genes was observed, which was possibly mediated by a higher activity of hypoxia-inducible factor 1α. In addition, the results of the gene set enrichment analysis suggested that MB contributed to fatty acid transport and turnover. MB-positive, wild-type-p53 LNCaP cells also exhibited increased expression of p53 target genes involved in cell cycle checkpoint control and prevention of cell migration. MB-positive cells expressing mutant p53 exhibited upregulation of genes associated with prolonged cancer cell viability and motility. Therefore, it was hypothesized that these transcriptomic differences may result from MB-mediated generation of nitric oxide or reactive oxygen species, thus employing established enzymatic activities of the globin. In summary, the transcriptome comparisons identified potential molecular functions of MB in carcinogenesis by highlighting the interaction of MB with key metabolic and regulatory processes. CA extern

Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by co-immunoprecipitation. The tandem affinity purification (TAP) method allows an unbiased screen of potential interaction partners of bacterial effectors in a physiological approach: target cells can be infected with a bacterial strain harboring the TAP-tagged bacterial effector protein which is translocated in the host similar as under physiological infection conditions. No transfection and overexpression of the bacterial protein in the eukaryotic host are needed. Therefore, also host target cells not easy to transfect can be analyzed by this method. Moreover, the two consecutive affinity tags Calmodulin-Binding-Peptide (CBP) and Streptavidin-Binding-Peptide (SBP) fused to the translocated bacterial protein allow an outstanding clear purification of protein complexes formed between the bacterial protein of interest and host cell proteins with less occurrence of contaminants. Mass spectrometry allows an unbiased identification of interacting eukaryotic proteins.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.