128 research outputs found
Sorting of lysosomal proteins
AbstractLysosomes are composed of soluble and transmembrane proteins that are targeted to lysosomes in a signal-dependent manner. The majority of soluble acid hydrolases are modified with mannose 6-phosphate (M6P) residues, allowing their recognition by M6P receptors in the Golgi complex and ensuing transport to the endosomal/lysosomal system. Other soluble enzymes and non-enzymatic proteins are transported to lysosomes in an M6P-independent manner mediated by alternative receptors such as the lysosomal integral membrane protein LIMP-2 or sortilin. Sorting of cargo receptors and lysosomal transmembrane proteins requires sorting signals present in their cytosolic domains. These signals include dileucine-based motifs, DXXLL or [DE]XXXL[LI], and tyrosine-based motifs, YXXØ, which interact with components of clathrin coats such as GGAs or adaptor protein complexes. In addition, phosphorylation and lipid modifications regulate signal recognition and trafficking of lysosomal membrane proteins. The complex interaction of both luminal and cytosolic signals with recognition proteins guarantees the specific and directed transport of proteins to lysosomes
Acute renal proximal tubule alterations during induced metabolic crises in a mouse model of glutaric aciduria type 1
AbstractThe metabolic disorder glutaric aciduria type 1 (GA1) is caused by deficiency of the mitochondrial glutaryl-CoA dehydrogenase (GCDH), leading to accumulation of the pathologic metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in blood, urine and tissues. Affected patients are prone to metabolic crises developing during catabolic conditions, with an irreversible destruction of striatal neurons and a subsequent dystonic–dyskinetic movement disorder. The pathogenetic mechanisms mediated by GA and 3OHGA have not been fully characterized. Recently, we have shown that GA and 3OHGA are translocated through membranes via sodium-dependent dicarboxylate cotransporter (NaC) 3, and organic anion transporters (OATs) 1 and 4. Here, we show that induced metabolic crises in Gcdh−/− mice lead to an altered renal expression pattern of NaC3 and OATs, and the subsequent intracellular GA and 3OHGA accumulation. Furthermore, OAT1 transporters are mislocalized to the apical membrane during metabolic crises accompanied by a pronounced thinning of proximal tubule brush border membranes. Moreover, mitochondrial swelling and increased excretion of low molecular weight proteins indicate functional tubulopathy. As the data clearly demonstrate renal proximal tubule alterations in this GA1 mouse model during induced metabolic crises, we propose careful evaluation of renal function in GA1 patients, particularly during acute crises. Further studies are needed to investigate if these findings can be confirmed in humans, especially in the long-term outcome of affected patients
Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype
Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.This work was funded by the European Union’s Horizon 2020 research and innovation programme (BATCure, grant No. 666918 to DLM, JPB, SEM, TB and SS). JPB is funded by the Agencia Estatal de Investigación (PID2019-105699RB-I00/ AEI / 10.13039/501100011033 and RED2018-102576-T), Plan Nacional sobre Drogas (2020I028), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces. SS was funded by a grant from the Mila’s Miracle Foundation. TB was supported by German Research Council (DFG) grant FOR2625. SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards laboratory and office space. We acknowledge Marcella Cesana for providing the TFEB virus. Graphical abstract was created using BioRender.com
Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite
Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1). Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA). Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion
Disruption of the Autophagy-Lysosome Pathway Is Involved in Neuropathology of the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis
Variant late-infantile neuronal ceroid lipofuscinosis, a fatal lysosomal storage disorder accompanied by regional atrophy and pronounced neuron loss in the brain, is caused by mutations in the CLN6 gene. CLN6 is a non-glycosylated endoplasmic reticulum (ER)-resident membrane protein of unknown function. To investigate mechanisms contributing to neurodegeneration in CLN6 disease we examined the nclf mouse, a naturally occurring model of the human CLN6 disease. Prominent autofluorescent and electron-dense lysosomal storage material was found in cerebellar Purkinje cells, thalamus, hippocampus, olfactory bulb and in cortical layer II to V. Another prominent early feature of nclf pathogenesis was the localized astrocytosis that was evident in many brain regions and the more widespread microgliosis. Expression analysis of mutant Cln6 found in nclf mice demonstrated synthesis of a truncated protein with a reduced half-life. Whereas the rapid degradation of the mutant Cln6 protein can be inhibited by proteasomal inhibitors, there was no evidence for ER stress or activation of the unfolded protein response in various brain areas during postnatal development. Age-dependent increases in LC3-II, ubiquitinated proteins, and neuronal p62-positive aggregates were observed, indicating a disruption of the autophagy-lysosome degradation pathway of proteins in brains of nclf mice, most likely due to defective fusion between autophagosomes and lysosomes. These data suggest that proteasomal degradation of mutant Cln6 is sufficient to prevent the accumulation of misfolded Cln6 protein, whereas lysosomal dysfunction impairs constitutive autophagy promoting neurodegeneration
N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells
The cellular prion protein (PrPC) plays a fundamental role in prion disease. PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrPC is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrPC and also replaced the GPI-anchor of PrPC by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrPC in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrPC. Exchange of the PrPC GPI-anchor for the one of Thy-1 redirects PrPC to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrPC, with the GPI-anchor being dominant over N-glycans
Organic anion transporters OAT1 and OAT4 mediate the high affinity transport of glutarate derivatives accumulating in patients with glutaric acidurias
- …