No abstract

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10-to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuronspecific Na + /K + -ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn 879 and Trp 880 is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.NMR | computational modeling | abnormal protein-protein interaction in synapse | hyperexcitotoxicity | protein-protein interaction inhibitors

Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC) play an important role in NF-jB activation. However, the regulation of linear ubiquitin chain generation by LUBAC is not well characterized. Here, we identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear linkage specificity (OTULIN/Gumby/ FAM105B) and cylindromatosis (CYLD) that can cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP interacts with both CYLD and OTULIN even in unstimulated cells. The interaction of CYLD and OTULIN with HOIP synergistically suppresses LUBACmediated linear polyubiquitination and NF-jB activation. Moreover, introduction of a HOIP mutant unable to bind either deubiquitinase into HOIP-null cells augments the activation of NF-jB by TNF-a stimulation. Thus, the interactions between these two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling the extent of TNF-a-induced NF-jB activation in cells by fine-tuning the generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling pathway activation by LUBAC. Our observations provide molecular insights into the roles of ligase-deubiquitinase interactions in regulating molecular events resulting from linear ubiquitin conjugation.