Dataset Information

Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1.

ABSTRACT: Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1.

SUBMITTER: Spooner E

PROVIDER: S-EPMC3572064 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1.

Spooner Ellen E McLaughlin Brooke M BM Lepow Talya T Durns Tyler A TA Randall Justin J Upchurch Cameron C Miller Katherine K Campbell Erin M EM Fares Hanna H

PloS one 20130213 2

Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRP ...[more]

PMID: 23418601

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Project description:TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.

Project description:BACKGROUND:Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. METHODS:We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. RESULTS:We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases. CONCLUSIONS:The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. TRIAL REGISTRATION:ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.

Dataset Information

Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1.

Publications

Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1.

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