NeuroMab

Abnormal insoluble ubiqitinated protein aggregates are found in the brains of Huntington's disease (HD) patients and in mice transgenic for the HTT mutation. Here, we describe the earliest stages of visible NII formation in brains of R6/2 mice killed between 2 and 6 weeks of age. We found that huntingtin-positive aggregates formed rapidly (within 24-48 hours) in a spatiotemporal manner similar to that we described previously for ubiquitinated inclusions. However, in most neurons, aggregates are not ubiquitinated when they first form. It has always been assumed that mutant huntingtin is recognised as 'foreign' and consequently ubiquitinated and targeted for degradation by the ubiquitin-proteasome system pathway. Our data, however, suggest that aggregation and ubiquitination are separate processes, and that mutant huntingtin fragment is not recognized as 'abnormal' by the ubiquitin-proteasome system before aggregation. Rather, mutant Htt appears to aggregate before it is ubiquitinated, and then either aggregated huntingtin is ubiquitinated or ubiquitinated proteins are recruited into aggregates. Our findings have significant implications for the role of the ubiquitin-proteasome system in the formation of aggregates, as they suggest that this system is not involved until after the first aggregates form.

Antibodies are important tools with diverse uses in biomedical research. However, open access to reliable sources of well-characterized antibodies with unambiguous molecular identities remains an obstacle to research transparency and reproducibility. We propose here a community shift towards open-source antibodies, analogous to open-source computer software. The tenets of such antibodies are that 1) they are available to researchers in a ready to use form, 2) the renewable source of the antibody (e.g., hybridoma cells or plasmid) is also widely available ensuring reproducible and cost-effective access to the same antibody, and 3) the antibody sequence is publicly available. With these criteria met, the antibody can be widely used with the transparent assurance associated with a molecularly defined reagent, and the code can be edited to generate antibody variants to meet researchers' specific needs. We (the UC Davis/NIH NeuroMab Facility, the Development Studies Hybridoma Bank, and Addgene) have established a consortium to provide open-source access to a large collection of well characterized antibodies. As open-source software has benefitted both users and developers, we suggest open-source antibodies will have a similar positive impact on antibody based biomedical research. We encourage funding agencies to support initiatives to expand access to open-source antibody resources, and researchers to both utilize and to contribute to them, with a goal of enabling more reliable and cost-effective pursuit of research.

Eighty-five percent of the human proteome has at least one interacting monoclonal antibody. These molecules penetrate the cytoplasm poorly and are very often non-functional within the cell. Analysis of antibody variable domains and characterisation of forty-five single-chain variable fragment (scFv) intrabodies expressed in human cells indicated charge to have the greatest impact on solubility. We created new interdomain linkers, optimised scFv domain orientation and found an optimisable charge discrepancy between variable heavy framework and CDR sites. When applied to reduce the search space and rank the products of AI-led inverse folding this creates a single highly soluble, abundant and stable intrabody with parent antibody epitope recognition. Over six hundred intrabody sequences are presented targeting sixty cytoplasmic proteins with linear, conformational, post-translational modification or oligomer specificity. Interactions were validated for p53, α-synuclein, SOD1, polyQ, FUS/TLS, UCHL1 and GFP. Here we show reliable repurposing of the sequenced antibody interactome inside the cell.