Oligomerization of ZFYVE27 (Protrudin) Is Necessary to Promote Neurite Extension

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27ΔHR3 failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27ΔHR3 was co-expressed with wild-type ZFYVE27 (ZFYVE27WT), it exerted a dominant negative effect on ZFYVE27WT as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions

Transcriptional and Post-Transcriptional Regulation of SPAST, the Gene Most Frequently Mutated in Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70–80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3′-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP

Spastin oligomerizes into a hexamer and the mutant spastin (E442Q) redistribute the wild-type spastin into filamentous microtubule

Spastin, a member of the ATPases associated with various cellular activities (AAA) family of proteins, is the most frequently mutated in hereditary spastic paraplegia. The defining feature of the AAA proteins is a structurally conserved AAA domain which assembles into an oligomer. By chemical cross linking and gel filtration chromatography, we show that spastin oligomerizes into a hexamer. Furthermore, to gain a comprehensive overview of the oligomeric structure of spastin, we generated a structural model of the AAA domain of spastin using template structure of VPS4B and p97/VCP. The generated model of spastin provided us with a framework to classify the identified missense mutations in the AAA domain from hereditary spastic paraplegia patients into different structural/functional groups. Finally, through co-localization studies in mammalian cells, we show that E442Q mutant spastin acts in a dominant negative fashion and causes redistribution of both wild-type spastin monomer and spastin interacting protein, RTN1 into filamentous microtubule bundles

Expansion of mutation spectrum, determination of mutation cluster regions and predictive structural classification of SPAST mutations in hereditary spastic paraplegia

The SPAST gene encoding for spastin plays a central role in the genetically heterogeneous group of diseases termed hereditary spastic paraplegia (HSP). In this study, we attempted to expand and refine the genetic and phenotypic characteristics of SPAST associated HSP by examining a large cohort of HSP patients/families. Screening of 200 unrelated HSP cases for mutations in the SPAST gene led to detection of 57 mutations (28.5%), of which 47 were distinct and 29 were novel mutations. The distribution analysis of known SPAST mutations over the structural domains of spastin led to the identification of several regions where the mutations were clustered. Mainly, the clustering was observed in the AAA (ATPases associated with diverse cellular activities) domain; however, significant clustering was also observed in the MIT (microtubule interacting and trafficking), MTBD (microtubule-binding domain) and an N-terminal region (228–269 residues). Furthermore, we used a previously generated structural model of spastin as a framework to classify the missense mutations in the AAA domain from the HSP patients into different structural/functional groups. Our data also suggest a tentative genotype–phenotype correlation and indicate that the missense mutations could cause an earlier onset of the disease

Repeated ER-endosome contacts promote endosome translocation and neurite outgrowth

The main organelles of the secretory and endocytic pathways--the endoplasmic reticulum (ER) and endosomes, respectively--are connected through contact sites whose numbers increase as endosomes mature. One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase, whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein or mediate endosome fission. Cholesterol transfer and Ca(2+) exchange have been proposed as additional functions of such sites. However, the compositions, activities and regulations of ER-endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth, forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE-ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER-LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth