A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules

Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small-molecule–protein interactions

Gruenewald Family Collection 1959-1967

Family trees, tree certificates for memorial plantings in Israel, artworks, photograph of a 'synagoga' statue, engraving of Breslau (Wrocław); drawings by Helmuth Nathan, H. Weyl?, and one other artist.Linked to online manifestationdigitize

Investigating the role of orphan GPR50 in normal brain function and mental illness

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signaling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. G protein-coupled receptor 50 (GPR50) is one such orphan, and its exact role is yet unknown. There is however emerging functional and genetic evidence suggesting a function for GPR50 in psychiatric illness and lipid metabolism. It was hypothesised that investigating GPR50’s protein-protein interactions would lead to a greater understanding of the role of GPR50 in normal brain functioning and in mental illness. Putative protein interactors were initially isolated by a yeast two-hybid study and were further tested here. To address GPR50’s links to mental illness, the GPR50∆502-505 deletion variant associated with mood disorders was also investigated. To test this hypothesis I sought to confirm some of the key yeast two-hybrid interactions. Using co-immunoprecipitation and immunocytochemistry the interaction of GPR50 with reticulon family members Nogo-A, Nogo-C and RTN3, and with cell-cell adhesion molecule CDH8 and lipid-associated protein ABCA2 were validated. In order to identify the location of interactions, subcellular fractionation of mouse brain and rt-PCR and immunohistochemistry in developing and adult mouse brain were performed. GPR50 and several interactors were found to be enriched at the synapse by subcellular fractionation of whole adult brain, and at embryonic day 18 (E18) and 5 weeks by rt-PCR. Colocalisation of GPR50 and interactors was found in the amygdala, hypothalamus, cortex and specific brain stem nuclei by immunohistochemistry. The discovery of GPR50 expression in noradrenergic, serotonergic and dopaminergic nuclei in the adult brain stem suggests a further role for GPR50 in neurotransmitter signaling and stress. To investigate the function of GPR50 two assays were performed that measure processes which are known to be affected by Nogo and RTN3: The first assay was a neurite outgrowth assay in Neuroscreen-1 cells, a PC12 cell clone. A significant increase in neurite length was detected after transient overexpression of GPR50 and this effect was increased in the GPR50∆502-505/T532A variant. Additionally GPR50-overexpression resulted in an increase in filopodia formation suggesting a role in actin dynamics. As a second functional assay in vitro BACE1 activity assays were performed in HEK293 cells. GPR50 but not GPR50∆502-505/T532A overexpression resulted in a significant increase in BACE1 activity. Lastly a final series of pilot experiments were performed to gain insight into the secondary structure of the C-terminal domain and the effects of the polymorphisms on structure. The 35kDa GPR50 C-terminal domain was purified and Circular Dichroism studies indicated a predominantly unstructured protein with increased a- helical content in the GPR50∆502-505 variant. The results in this thesis indicate a role for GPR50 in neuronal development and synaptic functioning. The results also strengthen an association with major mental illness, with links to several disease mechanisms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Here, there, and above : In memory of Hede /

A collection of various short stories, written 1952-1975 mostly relating to spiritual Jewish life, by Gruenewald and his wife Hede for their congregational bulletin

Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis