Functional multimerization of mucolipin channel proteins

MCOLN1 encodes mucolipin-1 (TRPML1), a member of the transient receptor potential TRPML subfamily of channel proteins. Mutations in MCOLN1 cause mucolipidosis-type IV (MLIV), a lysosomal storage disorder characterized by severe neurologic, ophthalmologic, and gastrointestinal abnormalities. Along with TRPML1, there are two other TRPML family members, mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3). In this study, we used immunocytochemical analysis to determine that TRPML1, TRPML2, and TRPML3 co-localize in cells. The multimerization of TRPML proteins was confirmed by co-immunoprecipitation and Western blot analysis, which demonstrated that TRPML1 homo-multimerizes as well as hetero-multimerizes with TRPML2 and TRPML3. MLIV-causing mutants of TRPML1 also interacted with wild-type TRPML1. Lipid bilayer re-constitution of in vitro translated TRPML2 and TRPML3 confirmed their cation channel properties with lower single channel conductance and higher partial permeability to anions as compared to TRPML1. We further analyzed the electrophysiological properties of single channel TRPML hetero-multimers, which displayed functional differences when compared to individual TRPMLs. Our data shows for the first time that TRPMLs form distinct functional channel complexes. Homo- and hetero-multimerization of TRPMLs may modulate channel function and biophysical properties, thereby increasing TRPML functional diversity.Fil: Curcio Morelli, Cyntia. Harvard Medical School; Estados UnidosFil: Zhang, Peng. Massachusetts General Hospital East; Estados UnidosFil: Venugopal, Bhuvarahamurthy. Harvard Medical School; Estados UnidosFil: Charles, Florie A.. Harvard Medical School; Estados UnidosFil: Browning, Marsha F.. Harvard Medical School; Estados UnidosFil: Cantiello, Horacio Fabio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Massachusetts General Hospital East; Estados UnidosFil: Slaugenhaupt, Susan A.. Harvard Medical School; Estados Unido

Neurologic, Gastric, and Opthalmologic Pathologies in a Murine Model of Mucolipidosis Type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene, which encodes the 65-kDa protein mucolipin-1. The most common clinical features of patients with MLIV include severe mental retardation, delayed motor milestones, ophthalmologic abnormalities, constitutive achlorhydria, and elevated plasma gastrin levels. Here, we describe the first murine model for MLIV, which accurately replicates the phenotype of patients with MLIV. The Mcoln1−/− mice present with numerous dense inclusion bodies in all cell types in brain and particularly in neurons, elevated plasma gastrin, vacuolization in parietal cells, and retinal degeneration. Neurobehavioral assessments, including analysis of gait and clasping, confirm the presence of a neurological defect. Gait deficits progress to complete hind-limb paralysis and death at age ∼8 mo. The Mcoln1−/− mice are born in Mendelian ratios, and both male and female Mcoln1−/− mice are fertile and can breed to produce progeny. The creation of the first murine model for human MLIV provides an excellent system for elucidating disease pathogenesis. In addition, this model provides an invaluable resource for testing treatment strategies and potential therapies aimed at preventing or ameliorating the abnormal lysosomal storage in this devastating neurological disorder

Deubiquitination of type 2 iodothyronine deiodinase by von Hippel–Lindau protein–interacting deubiquitinating enzymes regulates thyroid hormone activation

The type 2 iodothyronine deiodinase (D2) is an integral membrane ER-resident selenoenzyme that activates the pro-hormone thyroxine (T4) and supplies most of the 3,5,3′-triiodothyronine (T3) that is essential for brain development. D2 is inactivated by selective conjugation to ubiquitin, a process accelerated by T4 catalysis and essential for the maintenance of T3 homeostasis. A yeast two-hybrid screen of a human-brain library with D2 as bait identified von Hippel–Lindau protein–interacting deubiquitinating enzyme-1 (VDU1). D2 interaction with VDU1 and VDU2, a closely related deubiquitinase, was confirmed in mammalian cells. Both VDU proteins colocalize with D2 in the ER, and their coexpression prolongs D2 half-life and activity by D2 deubiquitination. VDU1, but not VDU2, is markedly increased in brown adipocytes by norepinephrine or cold exposure, further amplifying the increase in D2 activity that results from catecholamine-stimulated de novo synthesis. Thus, deubiquitination regulates the supply of active thyroid hormone to brown adipocytes and other D2-expressing cells

THE THYROID HORMONE INACTIVATING DEIODINASE FUNCTIONS AS HOMODIMER

International audienceThe type 3 deiodinase (D3) inactivates thyroid hormone action by catalyzing tissue-specific inner ring de- iodination, predominantly during embryonic development. D3 has gained much attention as a player in the euthyroid sick syndrome, given its robust reactivation during injury and/or illness. While much of the structure biology of the deiodinases is derived from studies with D2, a dimeric endoplasmic-reticulum obligatory activating deiodinase, little is known about the holostructure of the plasma membrane-resident D3, the deiodinase capable of thyroid hormone inactivation. Here we used fluorescence resonance energy transfer (FRET) in live cells to demonstrate that D3 exists as homodimer. While D3 homodimerized in its native state, minor heterodimerization was also observed between D3:D1 and D3:D2 in intact cells, the significance of which remains elusive. Incubation with 0.5-1.2 M urea resulted in loss of D3 homodimeri- zation as assessed by bioluminescence resonance energy transfer (BRET) and a proportional loss of en- zyme activity, to a maximum of ~50%. Protein modeling using a D2-based scaffold identified potential dimerization surfaces in the transmembrane and globular domains. Truncation of the transmembrane do- main (ΔD3) abrogated dimerization and deiodinase activity except when co-expressed with full-length catalytically inactive deiodinase, thus assembled as ΔD3:D3 dimer; thus the D3 globular domain also ex- hibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities