Random mutagenesis of the prokaryotic peptide transporter YdgR identifies potential periplasmic gating residues.

The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT(so), identifies Glu(56) and Arg(305) as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.

Expression of hypoxia-associated proteins in sporadic medullary thyroid cancer is associated with desmoplastic stroma reaction and lymph node metastasis and may indicate somatic mutations in the VHL gene.

Medullary thyroid carcinomas (MTCs) are mostly aggressive but slowly growing malignant tumours that metastasize early to loco-regional lymph nodes. Desmoplastic stroma reaction is a strong risk factor associated with lymph node metastases. We evaluated immunohistochemically the expression of two hypoxia-associated proteins, carbonic anhydrase IX (CAIX) and hypoxia-induced factor 1α (HIF1α), and ki-67, intercellular matrix adhesion molecule E-cadherin and the stroma remodelling marker tenascin C in a series of 100 sporadic MTCs and corresponding lymph node metastases, if present. Moderate to strong expression of CAIX was seen in 53 cases, and of HIF1α in 51 cases, showing a strong correlation (p < 0.001; Spearman's coefficient of correlation, 0.59). Expression correlated with the degree of desmoplasia (pCAIX = 0.001 and pHIF1α = 0.001), with tenascin C expression (pCAIX = 0.001,pHIF1α = 0.038), with the ki-67 proliferation index (pCAIX = 0.001, pHIF1α = 0.001) and with the presence of lymph node metastases (pCAIX < 0.001 and pHIF1α = 0.007). The absence of membranous E-cadherin staining was significantly associated with the grade of desmoplasia, tenascin expression and lymph node metastases(p ≤ 0.05) but not with ki67 proliferation index or expression of hypoxia-associated factors. Expression of hypoxia-associated proteins was in most cases identical between primary tumours and lymph node metastases.Two cases showed strong uniform expression of CAIX and HIF1α in the primary tumour as well as in the lymph node metastases, and sequencing revealed mutations in the coding regions of the Von-Hippel­Lindau gene (VHL ).Our findings suggest that despite of the fact that MTCs have only slowly growth, tumour hypoxia plays an important role in the development of loco-regional metastases. Since traditional cytotoxic chemotherapy has only little effect on MTCs, targeting hypoxia-associated and -regulated proteins might be of benefit for patients.

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.

Apolipoprotein E receptors are required for reelin-induced proteasomal degradation of the neuronal adaptor protein Disabled-1.

The cytoplasmic adaptor protein Disabled-1 (Dab1) is necessary for the regulation of neuronal positioning in the developing brain by the secreted molecule Reelin. Binding of Reelin to the neuronal apolipoprotein E receptors apoER2 and very low density lipoprotein receptor induces tyrosine phosphorylation of Dab1 and the subsequent activation or relocalization of downstream targets like phosphatidylinositol 3 (PI3)-kinase and Nckbeta. Disruption of Reelin signaling leads to the accumulation of Dab1 protein in the brains of genetically modified mice, suggesting that Reelin limits its own action in responsive neurons by down-regulating the levels of Dab1 expression. Here, we use cultured primary embryonic neurons as a model to demonstrate that Reelin treatment targets Dab1 for proteolytic degradation by the ubiquitin-proteasome pathway. We show that tyrosine phosphorylation of Dab1 but not PI3-kinase activation is required for its proteasomal targeting. Genetic deficiency in the Dab1 kinase Fyn prevents Dab1 degradation. The Reelin-induced Dab1 degradation also depends on apoER2 and very low density lipoprotein receptor in a gene-dose dependent manner. Moreover, pharmacological blockade of the proteasome prevents the formation of a proper cortical plate in an in vitro slice culture assay. Our results demonstrate that signaling through neuronal apoE receptors can activate the ubiquitin-proteasome machinery, which might have implications for the role of Reelin during neurodevelopment and in the regulation of synaptic transmission.