Tubby-like Proteins Research Articles

Interaction between the Photoreceptor-Specific Tubby-like Protein 1 and the Neuronal-Specific GTPase Dynamin-1

Tubby-like proteins (TULPs) are a family of four proteins, two of which have been linked to neurosensory disease phenotypes. TULP1 is a photoreceptor-specific protein that is mutated in retinitis pigmentosa, an inherited retinal disease characterized by the degeneration of rod and cone photoreceptor cells. To investigate the function of TULP1 in maintaining the health of photoreceptors, the authors sought the identification of interacting proteins. Immunoprecipitation from retinal lysates, followed by liquid chromatography tandem mass spectrometry and in vitro binding assays, were used to identify TULP1 binding partners. RT-PCR was performed on total RNA from wild-type mouse retina to identify the Dynamin-1 isoform expressed in the retina. Immunocytochemistry was used to determine the localization of TULP1 and Dynamin-1 in photoreceptor cells. Electroretinography (ERG) and light microscopy were used to phenotype tulp1-/- mice at a young age. Immunoprecipitation from retinal lysate identified Dynamin-1 as a possible TULP1 binding partner. GST pull-down assays further supported an interaction between TULP1 and Dynamin-1. In photoreceptor cells, Dynamin-1 and TULP1 colocalized primarily to the outer plexiform layer, where photoreceptor terminals synapse on second-order neurons and, to a lesser extent, to the inner segments, where polarized protein translocation occurs. ERG analyses in young tulp1-/- mice indicated a decreased b-wave at ages when the retina retained a full complement of photoreceptor cells. These data indicated that TULP1 interacts with Dynamin-1 and suggested that TULP1 is involved in the vesicular trafficking of photoreceptor proteins, both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation to the outer segment. These results also raised the possibility that normal synaptic function requires TULP1, and they motivate a closer look at synaptic architecture in the developing tulp1-/- retina.

Defective carbohydrate metabolism in mice homozygous for the tubby mutation

Tub is a member of a small gene family, the tubby-like proteins (TULPs), with predominant expression in neurons. Mice carrying a mutation in Tub develop retinal and cochlear degeneration as well as late-onset obesity with insulin resistance. During behavioral and metabolic testing, we found that homozygous C57BL/6J-Tub(tub) mice have a lower respiratory quotient than C57BL/6J controls before the onset of obesity, indicating that tubby homozygotes fail to activate carbohydrate metabolism and instead rely on fat metabolism for energy needs. In concordance with this, tubby mice show higher excretion of ketone bodies and accumulation of glycogen in the liver. Quantitation of liver mRNA levels shows that, during the transition from light to dark period, tubby mice fail to induce glucose-6-phosphate dehydrogenase (G6pdh), the rate-limiting enzyme in the pentose phosphate pathway that normally supplies NADPH for de novo fatty acid synthesis and glutathione reduction. Reduced G6PDH protein levels and enzymatic activity in tubby mice lead accordingly to lower levels of NADPH and reduced glutathione (GSH), respectively. mRNA levels for the lipolytic enzymes acetyl-CoA synthetase and carnitine palmitoyltransferase are increased during the dark cycle and decreased during the light period, and several citric acid cycle genes are dysregulated in tubby mice. Examination of hypothalamic gene expression showed high levels of preproorexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. We hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the moderate obesity observed in tubby mice.

Abnormal cholinergic and GABAergic vascular innervation in the hypothalamic arcuate nucleus of obese tub/tub mice

Tubby and tubby-like proteins (TULPs) are encoded by members of a small gene family. An autosomal recessive mutation in the mouse tub gene leads to blindness, deafness, and maturity-onset obesity. The mechanisms by which the mutation causes the obesity syndrome has not been established. We compared obese tub/tub mice and their lean littermates in order to find abnormalities within the mediobasal hypothalamus, a region intimately associated with the regulation of body weight. Using an antiserum to the vesicular acetylcholine transporter (VAChT), a marker for cholinergic neurons, many unusually large VAChT-immunoreactive (-ir) nerve terminals, identified by colocalization with the synaptic vesicle protein synaptophysin, were demonstrated in the hypothalamic arcuate nucleus of obese tub/tub mice. Double-labeling showed that VAChT-ir nerve endings also contained glutamic acid decarboxylase (GAD), a marker for gamma-aminobutyric acid (GABA) neurons. The VAChT- and GAD-ir nerve terminals were in close contact with blood vessels, identified with antisera to platelet endothelial cell adhesion molecule-1 (PECAM; also called CD31), laminin, smooth muscle actin (SMA), and glucose transporter-1 (GLUT1). Such large cholinergic and GABAergic nerve terminals surrounding blood vessels were not seen in the arcuate nucleus of lean tub/+ mice. The presence of abnormal cholinergic/GABAergic vascular innervation in the arcuate nucleus suggests that alterations in this region, which contains neurons that receive information from the periphery and which relays information about the energy status to other parts of the brain, may be central in the development of the obese phenotype in animals with an autosomal recessive mutation in the tub gene.

Molecular analyses of the Arabidopsis TUBBY-like protein gene family.

In mammals, TUBBY-like proteins play an important role in maintenance and function of neuronal cells during postdifferentiation and development. We have identified a TUBBY-like protein gene family with 11 members in Arabidopsis, named AtTLP1-11. Although seven of the AtTLP genes are located on chromosome I, no local tandem repeats or gene clusters are identified. Except for AtTLP4, reverse transcription-PCR analysis indicates that all these genes are expressed in various organs in 6-week-old Arabidopsis. AtTLP1, 2, 3, 6, 7, 9, 10, and 11 are expressed ubiquitously in all the organs tested, but the expression of AtTLP5 and 8 shows dramatic organ specificity. These 11 family members share 30% to 80% amino acid similarities across their conserved C-terminal tubby domains. Unlike the highly diverse N-terminal region of animal TUBBY-like proteins, all AtTLP members except AtTLP8 contain a conserved F-box domain (51-57 residues). The interaction between AtTLP9 and ASK1 (Arabidopsis Skp1-like 1) is confirmed via yeast (Saccharomyces cerevisiae) two-hybrid assays. Abscisic acid (ABA)-insensitive phenotypes are observed for two independent AtTLP9 mutant lines, whereas transgenic plants overexpressing AtTLP9 are hypersensitive to ABA. These results suggest that AtTLP9 may participate in the ABA signaling pathway.

Tubby proteins: the plot thickens.

The tubby mouse, which shows late-onset obesity and neurosensory deficits, arises from a mutation in the Tub gene. Tub shares homology with the genes for tubby-like proteins Tulp1, Tulp2 and Tulp3. Ablation of Tub, Tulp1 or Tulp3 causes disease phenotypes that are indicative of their importance in nervous-system function and development. Despite this importance, the biochemical functions of tubby-like proteins are only now beginning to be understood. At present, data indicate that tubby-like proteins might function as heterotrimeric-G-protein-responsive intracellular signalling factors, although an array of data also implicates them in other processes.

Retinal degeneration caused by mutations in TULP1.

TULP1 is a member of a family of four proteins named TULPs for tubby-like proteins, defined by the highly conserved C-terminal half of their primary sequences (60 ¡ª 90% amino acid identity). This protein family includes TUB, TULP1, TULP2, and TULP3, all of which have different N-termini and different cellular expression patterns (North et al., 1997). Other than members of the TULP family, database searches do not reveal any significant homology with known proteins or functional motifs. Their physiological functions are unknown but two have been linked to photoreceptor degeneration. Mutations in the human TULPI gene were found to be a cause of retinitis pigmentosa (Hagstrom et al., 1998; Banerjee et al., 1998). Loss of Tulp1 function in mice, introduced by gene knockout, also causes photoreceptor degeneration (Hagstrom et al., 1999; Ikeda et al., 2000). A naturally occurring recessive null mutation in the tubby gene in mice causes photoreceptor and cochlear degeneration and adult-onset obesity (Ohlemiller et al., 1995).

Structure and expression patterns of Drosophila TULP and TUSP, members of the tubby-like gene family

Tubby is a mouse gene that may provide a model for adult-onset obesity in humans. It is a member of a four gene family in mammals that collectively encode the Tubby-like proteins (TULPs), putative transcription factors which share similar 260 amino acid ‘tubby domains’ at their C-termini. The mammalian genome also encodes distant relatives of TULPs, which have been called TUSPs (tubby domain superfamily proteins). We have characterized the transcription unit of the single Drosophila TULP homolog, analyzed the expression pattern of the Drosophila TULP and TUSP genes, and determined the evolutionary relationships between the Drosophila proteins and members of the tubby domain superfamily in other organisms. Interestingly, like its mammalian homologs, Drosophila TULP is principally expressed in the embryonic central and peripheral nervous systems. This suggests that mammalian and Drosophila TULPs may possess some conserved functional properties in the nervous system. The Drosophila TUSP gene is also expressed in the central nervous system and olfactory organ but in few other peripheral sensory organs.

Microtubule-associated protein 1A is a modifier of tubby hearing (moth1).

Once a mutation in the gene tub was identified as the cause of obesity, retinal degeneration and hearing loss in tubby mice, it became increasingly evident that the members of the tub gene family (tulps) influence maintenance and function of the neuronal cell lineage. Suggested molecular functions of tubby-like proteins include roles in vesicular trafficking, mediation of insulin signaling and gene transcription. The mechanisms through which tub functions in neurons, however, have yet to be elucidated. Here we report the positional cloning of an auditory quantitative trait locus (QTL), the modifier of tubby hearing 1 gene (moth1), whose wildtype alleles from strains AKR/J, CAST/Ei and 129P2/OlaHsd protect tubby mice from hearing loss. Through a transgenic rescue experiment, we verified that sequence polymorphisms in the neuron-specific microtubule-associated protein 1a gene (Mtap1a) observed in the susceptible strain C57BL/6J (B6) are crucial for the hearing-loss phenotype. We also show that these polymorphisms change the binding efficiency of MTAP1A to postsynaptic density molecule 95 (PSD95), a core component in the cytoarchitecture of synapses. This indicates that at least some of the observed polymorphisms are functionally important and that the hearing loss in C57BL/6J-tub/tub (B6-tub/tub) mice may be caused by impaired protein interactions involving MTAP1A. We therefore propose that tub may be associated with synaptic function in neuronal cells.

The tubby-like proteins, a family with roles in neuronal development and function.

The identification of a mutation at the tubby (Tub) locus, which causes obesity and neurosensory degeneration, led to the discovery of the tubby-like proteins (TULPs). Tub and the genes that encode three tubby-like proteins (TULP1- TULP3) form a novel, small gene family that plays an important role in maintenance and function of neuronal cells during development and post-differentiation. Although exploration of the molecular function of these genes is still in its infancy, recent biochemical studies have provided 'entry points' into pathways whose elucidation will further our understanding of TULP action. In addition, mRNA expression and translocation of the TUB protein have been shown to be regulated by thyroid hormone and by G-protein-coupled receptor signaling, respectively. These latter findings may help to link the cellular function of TUB to known mechanisms for energy homeostasis.

Implication of Tubby Proteins as Transcription Factors by Structure-Based Functional Analysis

Tubby-like proteins (TULPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TULPs can result in one or more of three disease phenotypes: obesity (from which the name “tubby” is derived), retinal degeneration, and hearing loss. These disease phenotypes indicate a vital role for tubby proteins; however, no biochemical function has yet been ascribed to any member of this protein family. A structure-directed approach was employed to investigate the biological function of these proteins. The crystal structure of the core domain from mouse tubby was determined at a resolution of 1.9 angstroms. From primarily structural clues, experiments were devised, the results of which suggest that TULPs are a unique family of bipartite transcription factors.

Implication of tubby proteins as transcription factors by structure-based functional analysis.

Tubby-like proteins (TULPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TULPs can result in one or more of three disease phenotypes: obesity (from which the name "tubby" is derived), retinal degeneration, and hearing loss. These disease phenotypes indicate a vital role for tubby proteins; however, no biochemical function has yet been ascribed to any member of this protein family. A structure-directed approach was employed to investigate the biological function of these proteins. The crystal structure of the core domain from mouse tubby was determined at a resolution of 1.9 angstroms. From primarily structural clues, experiments were devised, the results of which suggest that TULPs are a unique family of bipartite transcription factors.