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Yes / Incoherent neutron spectroscopy, in combination with dynamic light scattering, was used to investigate the effect of ligand binding on the center-of-mass self-diffusion and internal diffusive dynamics of Escherichia coli aspartate α-decarboxylase (ADC). The X-ray crystal structure of ADC in complex with the D-serine inhibitor was also determined, and molecular dynamics simulations were used to further probe the structural rearrangements that occur as a result of ligand binding. These experiments reveal that D-serine forms hydrogen bonds with some of the active site residues, that higher order oligomers of the ADC tetramer exist on ns–ms time-scales, and also show that ligand binding both affects the ADC internal diffusive dynamics and appears to further increase the size of the higher order oligomers. / TR acknowledges a PhD studentship jointly funded by the ILL and the Universität Hamburg (Federal Excellence Cluster Hamburg Centre for Ultrafast Imaging EXC 1074).

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This year the American Cancer Society predicts that 565,650 individuals will lose their life as a result of their battle with cancer. Due to its established roles in cancer and extracellular presentation, the Epidermal Growth Factor Receptor (EGFR) is an excellent target for anti-cancer drugs. It has been determined that matriptase and prostasin serine proteases are proteolytic regulators of EGFR membrane presentation, and downstream signaling. Currently, there are several drugs that target EGFR, but research continues in order to further understand drug-resistant EGFR. In cancer cell lines that exhibit both EGFR signaling and these proteases, proteolytic cleavage may be a mechanism of resistance to drugs that target the EGFR extracellular domain (ECD). The specific aim of this project was to determine which protease was direct! y responsible for EGFR cleavage and establish the precise cleavage site within the EGFR ECD. DNA corresponding to amino acid residues 336-505 of the EGFR ECD was cloned into the p-GEX-6P-I vector and expressed as a GST-fusion protein in E.coli cells. This fusion protein was isolated and purified by affinity chromatography. Purified GSTEGFR BCD fusion protein was mixed with prostasin and matriptase and evaluated for cleavage. No cleavage was detected using this method. Trypsin serine protease was used to ensure the cleavability of the GST-EGFR ECD. The GST-EGFR ECD fusion protein was found to be inappropriate for determining matriptase or prostasin cleavage sites, which are now being pursued by other means.

Yes / The mammalian X and Y chromosomes originated from a pair of ordinary autosomes. Over the past ~180 million years, the X and Y have become highly differentiated and now only recombine with each other within a short pseudoautosomal region. While the X chromosome broadly preserved its gene content, the Y chromosome lost ~92% of the genes it once shared with the X chromosome. PRSSLY is a Y-linked gene identified in only a few mammalian species that was thought to be acquired, not ancestral. However, PRSSLY's presence in widely divergent species-bull and mouse-led us to further investigate its evolutionary history. We discovered that PRSSLY is broadly conserved across eutherians and has ancient origins. PRSSLY homologs are found in syntenic regions on the X chromosome in marsupials and on autosomes in more distant animals, including lizards, indicating that PRSSLY was present on the ancestral autosomes but was lost from the X and retained on the Y in eutherian mammals. We found that across eutheria, PRSSLY's expression is testis-specific, and, in mouse, it is most robustly expressed in post-meiotic germ cells. The closest paralog to PRSSLY is the autosomal gene PRSS55, which is expressed exclusively in testes, involved in sperm differentiation and migration, and essential for male fertility in mice. Outside of eutheria, in species where PRSSLY orthologs are not Y-linked, we find expression in a broader range of somatic tissues, suggesting that PRSSLY has adopted a germ-cell-specific function in eutherians. Finally, we generated Prssly mutant mice and found that they are fully fertile but produce offspring with a modest female-biased sex ratio compared to controls. PRSSLY appears to be the first example of a gene that derives from the mammalian ancestral sex chromosomes that was lost from the X and retained on the Y. Although the function of PRSSLY remains to be determined, it may influence the sex ratio by promoting the survival or propagation of Y-bearing sperm. / The group of D.C.P. is supported by the Howard Hughes Medical Institute, the Whitehead Institute, and philanthropic gifts from Brit and Alexander d’Arbeloff, Authur W. and Carol Tobin Brill, and Charles Ellis.