RESUMO
The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP-Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Drosophila/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Transporte Axonal , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Feminino , Masculino , Microtúbulos/metabolismo , Neurônios/metabolismo , Fenótipo , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Transporte Proteico , Sinapses/metabolismoRESUMO
Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.
Assuntos
Ferritinas/química , Ferritinas/metabolismo , Ferro/metabolismo , Rhodospirillum rubrum/enzimologia , Rhodospirillum rubrum/metabolismo , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Cristalografia por Raios X , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Multimerização ProteicaRESUMO
Cytochrome P450s are b-heme-containing enzymes that are able to introduce oxygen atoms into a wide variety of organic substrates. They are extremely widespread in nature having diverse functions at both biochemical and physiological level. The genome of C. jejuni 81-176 encodes a single cytochrome P450 (Cj1411c) that has no close homologues. Cj1411c is unusual in its genomic location within a cluster involved in the biosynthesis of outer surface structures. Here we show that E. coli expressed and affinity-purified C. jejuni cytochrome P450 is lipophilic, containing one equivalent Cys-ligated heme. Immunoblotting confirmed the association of cytochrome P450 with membrane fractions. A Cj1411c deletion mutant had significantly reduced ability to infect human cells and was less able to survive following exposure to human serum when compared to the wild type strain. Phenotypically following staining with Alcian blue, we show that a Cj1411c deletion mutant produces significantly less capsular polysaccharide. This study describes the first known membrane-bound bacterial cytochrome P450 and its involvement in Campylobacter virulence.
Assuntos
Proteínas de Bactérias/genética , Infecções por Campylobacter/genética , Campylobacter jejuni/genética , Sistema Enzimático do Citocromo P-450/genética , Genes Bacterianos/genética , Proteínas de Bactérias/metabolismo , Infecções por Campylobacter/metabolismo , Campylobacter jejuni/metabolismo , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Células Epiteliais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Regulação para Cima/genética , VirulênciaRESUMO
This report presents a new approach to streak-seeding based on custom-designed silicon microtools. Experimental data show that the microtools produce similar results to the commonly used boar bristles. One advantage to using silicon is that it is rigid and can easily serve as an accurately calibrated end-effector on a micro-robotic system. Additionally, the fabrication technology allows the production of microtools of various shapes and sizes. A working prototype of an automatic streak-seeding system based on these microtools was built and successfully applied for protein crystallization.