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Introduction Hereditary spastic paraplegia 50 (SPG50; OMIM: 612936) is an ultrarare childhood-onset neurological disorder characterized by progressive spastic paraplegia, global Developmental delay, and subsequent intellectual disability, secondary microcephaly, early-onset epilepsy, [...], Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of- function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global Developmental delay, and subsequent intellectual disability, secondary microcephaly, and epilepsy. We preformed preclinical studies evaluating an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50 and describe in vitro studies that demonstrate transduction of patient-derived fibroblasts with AAV2/AP4M1, resulting in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1- KO mice were intrathecally (i.t.) injected with 5 * [10.sup.11], 2.5 * [10.sup.11], or 1.25 * [10.sup.11] vector genome (vg) doses of AAV9/AP4M1 at P7-P10 or P90. Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in WT mice, rats, and nonhuman primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1 * [10.sup.15] vg. Of note, similar degrees of minimal-to-mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future i.t. AAV studies. These preclinical results identify an acceptably safe and efficacious dose of i.t.- administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50.

The Golgi-localized, [gamma]-ear-containing, Arf (ADP-ribosylation factor)-binding (GGA) proteins are clathrin adaptors that mediate the sorting of transmembrane-cargo molecules at the trans-Golgi network and endosomes. Cargo proteins can be directed into the GGA pathway by at least two different types of sorting signals: acidic cluster-dileucine motifs and covalent modification by ubiquitin. The latter modification is recognized by the GGAs through binding to their GAT [GGA and TOM (target of Myb)] domain. Here we report the crystal structure of the GAT domain of human GGA3 in a 1:1 complex with ubiquitin at 2.8-A resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices [alpha]1 and [alpha]2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met231, Asp-244, Glu-246, Leu-247, Glu-250, and Leu-251. The GAT-binding surface on ubiquitin is a hydrophobic patch centered on Ile-44 that is also responsible for binding most other ubiquitin effectors. The ubiquitin-binding site observed in the crystal is distinct from the Rabaptin-5-binding site on helices [alpha]2 and [alpha]3 of the GAT domain. Mutational analysis and modeling of the ubiquitin-Rabaptin-5-GAT ternary complex indicates that ubiquitin and Rabaptin-5 can bind to the GAT domain at two different sites without any steric conflict. This ability highlights the GAT domain as a hub for interactions with multiple partners in trafficking. protein-protein interactions | structural biology | trafficking

The validity of a closed/open model of the myoglobin-carbon monoxide complex (MbCO) was analyzed using time-resolved Raman spectroscopy in recombinant human Mb and E7 mutants in which the distal histidine was replaced with other amino acids. The mutants with Fe-Co stretching frequency of 490/cm recover faster than those with Fe-Co stretching frequency of 507/cm. The open and closed MbCO forms were assigned a Fe-Co stretching frequency of 490/cm and 509/cm, respectively. As the E64 residues become mor hydrophobic, the Fe-Co stretching frequency becomes lower.

Splice Variants of the α Subunit of the G Protein G.sub.s Activate Both Adenylyl Cyclase and Calcium Channels PURIFIED PREPARATIONS OF G.sub.s, the stimulatory regulatory component of adenylyl cyclase, or [...]

An open question in cell biology is how the general intracellular transport machinery is adapted to perform specialized functions in polarized cells such as neurons. Here we illustrate this adaptation by elucidating a role for the ubiquitous small GTPase Ras-related protein in brain 5 (Rab5) in neuronal polarity. We show that inactivation or depletion of Rab5 in rat hippocampal neurons abrogates the somatodendritic polarity of the transferrin receptor and several glutamate receptor types, resulting in their appearance in the axon. This loss of polarity is not caused primarily by increased transport from the soma to the axon but rather by decreased retrieval from the axon to the soma. Retrieval is also dependent on the Rab5 effector Fused Toes (FTS)-Hook-FTS and Hook-interacting protein (FHIP) (FHF) complex, which interacts with the minus-end-directed microtubule motor dynein and its activator dynactin to drive a population of axonal retrograde carriers containing somatodendritic proteins toward the soma. These findings emphasize the importance of both biosynthetic sorting and axonal retrieval for the polarized distribution of somatodendritic receptors at steady state. [ABSTRACT FROM AUTHOR]