Recessive N-ethyl-N-nitrosourea (ENU)-induced mutations recovered at the fitness-1 (fit1) locus in mouse chromosome 7 cause hematopoietic abnormalities, growth retardation, and shortened life span, with varying severity of the defects in different alleles. Abnormal iron distribution and metabolism and frequent scoliosis have also been associated with an allele of intermediate severity (fit1 4R ). We report that fit1 4R , as well as the most severe fit1 5R allele, are nonsense point mutations in the mouse ortholog of the human phosphatidylinositol-binding clathrin assembly protein (PICALM) gene, whose product is involved in clathrin-mediated endocytosis. A variety of leukemias and lymphomas have been associated with translocations that fuse human PICALM with the putative transcription factor gene AF10. The Picalm fit1-5R and Picalm fit1-4R mutations are splice-donor alterations resulting in transcripts that are less abundant than normal and missing exons 4 and 17, respectively. These exon deletions introduce premature termination codons predicted to truncate the proteins near the N and C termini, respectively. No mutations in the genes encoding Picalm, clathrin, or components of the adaptor protein complex 2 (AP2) have been previously described in which the suite of disorders present in the Picalm fit1 mutant mice is apparent. These mutants thus provide unique models for exploring how the endocytic function of mouse Picalm and the transport processes mediated by clathrin and the AP2 complex contribute to normal hematopoiesis, iron metabolism, and growth.

Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington's disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ42P10) and compared the results with those obtained for the wild-type (NtQ22P10). Our results show that the aggregation pathway of NtQ22P10 is very different from that of NtQ42P10, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ42P10 are monomer and dimer. The divergent pathways ultimately result in NtQ22P10 fibrils that possess a packing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ42P10 fibrils present a better fit to the Perutz β-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.

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Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR) separate from their mutual binding. Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain α-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. By focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.