FAM83H and Autosomal Dominant Hypocalcified Amelogenesis Imperfecta

Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI; OMIM #130900) is a genetic disorder exhibiting severe hardness defects and reduced fracture toughness of dental enamel. While the condition is nonsyndromic, it can be associated with other craniofacial anomalies, such as malocclusions and delayed or failed tooth eruption. Truncation mutations in FAM83H (OMIM *611927) are hitherto the sole cause of ADHCAI. With human genetic studies, Fam83h knockout and mutation–knock-in mouse models indicated that FAM83H does not serve a critical physiologic function during enamel formation and suggested a neomorphic mutation mechanism causing ADHCAI. The function of FAM83H remains obscure. FAM83H has been shown to interact with various isoforms of casein kinase 1 (CK1) and keratins and to mediate organization of keratin cytoskeletons and desmosomes. By considering FAM83H a scaffold protein to anchor CK1s, further molecular characterization of the protein could gain insight into its functions. In this study, we characterized 9 kindreds with ADHCAI and identified 3 novel FAM83H truncation mutations: p.His437*, p.Gln459*, and p.Glu610*. Some affected individuals exhibited hypoplastic phenotypes, in addition to the characteristic hypocalcification enamel defects, which have never been well documented. Failed eruption of canines or second molars in affected persons was observed in 4 of the families. The p.Glu610* mutation was located in a gap area (amino acids 470 to 625) within the zone of previously reported pathogenic variants (amino acids 287 to 694). In vitro pull-down studies with overexpressed FAM83H proteins in HEK293 cells demonstrated an interaction between FAM83H and SEC16A, a protein component of the COP II complex at endoplasmic reticulum exit sites. The interaction was mediated by the middle part (amino acids 287 to 657) of mouse FAM83H protein. Results of this study significantly extended the phenotypic and genotypic spectrums of FAM83H-associated ADHCAI and suggested a role for FAM83H in endoplasmic reticulum–to–Golgi vesicle trafficking and protein secretion (dbGaP phs001491.v1.p1).

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Enamelin and Autosomal-dominant Amelogenesis Imperfecta

Critical Reviews in Oral Biology & Medicine ◽

10.1177/154411130301400602 ◽

2003 ◽

Vol 14 (6) ◽

pp. 387-398 ◽

Cited By ~ 92

Author(s):

J.C.-C. Hu ◽

Y. Yamakoshi

Keyword(s):

Autosomal Dominant ◽

Dental Enamel ◽

Severe Form ◽

Amelogenesis Imperfecta ◽

Post Translational Modifications ◽

Enamel Protein ◽

Enamel Proteins ◽

Temporal And Spatial ◽

Secretory Products

Dental enamel forms as a progressively thickening extracellular layer by the action of proteins secreted by ameloblasts. The most abundant enamel protein is amelogenin, which is expressed primarily from a gene on the X-chromosome (AMELX). The two most abundant non-amelogenin enamel proteins are ameloblastin and enamelin, which are expressed from the AMBN and ENAM genes, respectively. The human AMBN and ENAM genes are located on chromosome 4q13.2. The major secretory products of the human AMELX, AMBN, and ENAM genes have 175, 421, and 1103 amino acids, respectively, and are all post-translationally modified, secreted, and processed by proteases. Mutations in AMELX have been shown to cause X-linked amelogenesis imperfecta (AI), which accounts for 5% of AI cases. Mutations in ENAM cause a severe form of autosomal-dominant smooth hypoplastic AI that represents 1.5%, and a mild form of autosomal-dominant local hypoplastic AI that accounts for 27% of AI cases in Sweden. The discovery of mutations in the ENAM gene in AI kindreds proved that enamelin is critical for proper dental enamel formation and that it plays a role in human disease. Here we review how enamelin was discovered, what is known about enamelin protein structure, post-translational modifications, processing by proteases, and its potentially important functional properties such as its affinity for hydroxyapatite and influence on crystal growth in vitro. The primary structures of human, porcine, mouse, and rat enamelin are compared, and the human enamelin gene, its structure, chromosomal localization, temporal and spatial patterns of expression, and its role in the etiology of amelogenesis imperfecta are discussed.

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A Novel Missense Mutation (p.P52R) in Amelogenin Gene Causing X-linked Amelogenesis Imperfecta

Journal of Dental Research ◽

10.1177/154405910708600111 ◽

2007 ◽

Vol 86 (1) ◽

pp. 69-72 ◽

Cited By ~ 20

Author(s):

M. Kida ◽

Y. Sakiyama ◽

A. Matsuda ◽

S. Takabayashi ◽

H. Ochi ◽

...

Keyword(s):

Novel Mutation ◽

Dental Enamel ◽

Fluorescence Analysis ◽

Hereditary Disease ◽

Amelogenesis Imperfecta ◽

X Ray Diffraction ◽

Japanese Family ◽

X Ray ◽

Amelogenin Gene

Amelogenesis imperfecta (AI) is a hereditary disease with abnormal dental enamel formation. Here we report a Japanese family with X-linked AI transmitted over at least four generations. Mutation analysis revealed a novel mutation (p.P52R) in exon 5 of the amelogenin gene. The mutation was detected as heterozygous in affected females and as hemizygous in their affected father. The affected sisters exhibited vertical ridges on the enamel surfaces, whereas the affected father had thin, smooth, yellowish enamel with distinct widening of inter-dental spaces. To study the pathological cause underlying the disease in this family, we synthesized the mutant amelogenin p.P52R protein and evaluated it in vitro. Furthermore, we studied differences in the chemical composition between normal and affected teeth by x-ray diffraction analysis and x-ray fluorescence analysis. We believe that these results will greatly aid our understanding of the pathogenesis of X-linked AI.

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The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide.

The Journal of Cell Biology ◽

10.1083/jcb.104.6.1705 ◽

1987 ◽

Vol 104 (6) ◽

pp. 1705-1714 ◽

Cited By ~ 19

Author(s):

J Finidori ◽

L Rizzolo ◽

A Gonzalez ◽

G Kreibich ◽

M Adesnik ◽

...

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Signal Sequence ◽

In Vitro Translation ◽

Signal Peptidase ◽

Endoplasmic Reticulum Membrane ◽

Hydrophobic Region ◽

Amino Terminal ◽

Influenza Hemagglutinin

The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)

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Characterization of the kinase activity of a WNK4 protein complex

AJP Renal Physiology ◽

10.1152/ajprenal.00358.2009 ◽

2009 ◽

Vol 297 (3) ◽

pp. F685-F692 ◽

Cited By ~ 20

Author(s):

Robert Ahlstrom ◽

Alan S. L. Yu

Keyword(s):

Kinase Activity ◽

Genetic Disorder ◽

Signal Transduction Pathway ◽

Kinase Domain ◽

Full Length ◽

Hek293 Cells ◽

Pseudohypoaldosteronism Type Ii

Mutations in WNK4 protein kinase cause pseudohypoaldosteronism type II (PHAII), a genetic disorder that is characterized by renal NaCl and K+ retention leading to hypertension and hyperkalemia. Consistent with this, WNK4 is known to regulate several renal tubule transporters, including the NaCl cotransporter, NCC, and the K+ channel, ROMK, but the mechanisms are incompletely understood, and the role of the kinase activity in its actions is highly controversial. To assay WNK4 kinase activity, we have now succeeded in expressing and purifying full-length, enzymatically active WNK4 protein from HEK293 cells. We show that full-length wild-type WNK4 phosphorylates oxidative stress response kinase 1 (OSR1) and Ste20/SPS1-related proline/alanine-rich kinase (SPAK) in vitro. Introducing the PHAII-associated mutations, E559K, D561A, and Q562E, into our protein had no significant effect on this phosphorylation. We conclude that PHAII is unlikely to be caused by abnormal WNK4 kinase activity. We also made the intriguing observation that inactivating mutations of the WNK4 kinase domain did not completely abolish in vitro phosphorylation of OSR1/SPAK. Led by this, we identified a novel 40-kDa kinase that associates specifically with the COOH-terminal half of WNK4 and is able to phosphorylate both WNK4 and SPAK/OSR1. We suggest that this 40-kDa kinase functions in the WNK4 signal transduction pathway and may mediate some of the physiological actions attributed to WNK4.

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NSP-encoded reticulons, neuroendocrine proteins of a novel gene family associated with membranes of the endoplasmic reticulum

Journal of Cell Science ◽

10.1242/jcs.107.9.2403 ◽

1994 ◽

Vol 107 (9) ◽

pp. 2403-2416 ◽

Cited By ~ 2

Author(s):

H.J. van de Velde ◽

A.J. Roebroek ◽

N.H. Senden ◽

F.C. Ramaekers ◽

W.J. Van de Ven

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Gene Family ◽

Biochemical Characterization ◽

Smooth Endoplasmic Reticulum ◽

Dendritic Tree ◽

Cell Types ◽

In Vitro Translation ◽

Amino Terminal

The novel NSP gene was previously shown to encode, among a variety of neuroendocrine cell types, two 3′-overlapping transcripts, a 3.4 kb one for NSP-A (776 amino acids) and a 1.8 kb one for NSP-C (208 amino acids). The deduced proteins, which were predicted to possess distinct amino-terminal regions, appeared to exhibit some architectural resemblance to known neuroendocrine proteins. In this paper the biochemical characterization and subcellular localization of the two proteins is addressed. In vitro translation of NSP-A and -C RNA produced proteins of about 135 and 23 kDa, respectively. Proteins of similar molecular mass were also detected in immunoprecipitation and western blot analyses of neural and endocrine cells using specific anti-NSP-A or -C antisera; some heterogeneity of NSP-A was observed. NSP-A, but not NSP-C, appeared to be highly phosphorylated and preferentially on serine residues. In immunocytochemical studies, we demonstrated that NSP-A and -C are associated with the endoplasmic reticulum; NSP-A was found to co-localize with SERCA2b, a membrane-associated Ca(2+)-ATPase of the endoplasmic reticulum. In Purkinje cells, we found NSP-immunostaining in the perikaryon, the extensive dendritic tree and the axon, also suggesting association with the smooth endoplasmic reticulum. Biochemical studies of NSP-A provided evidence that NSP-A is strongly associated with microsomal membranes and analysis of deletion mutants of NSP-A revealed that the hydrophobic carboxy-terminal portion of the protein, which is also present in NSP-C, is critical for membrane binding. Through database searches, finally, we found two different NSP-related sequences, one in a sequenced region of human chromosome 19, and the second in a human, pancreatic islet-derived partial cDNA, suggesting that the NSP gene is the prototype of a larger gene family. The results of our studies seem to indicate that the NSP-encoded proteins are novel, membrane-anchored components of the endoplasmic reticulum for which we propose the name reticulons.

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Differential activation of parathyroid and renal Ca2+-sensing receptors underlies the renal phenotype in autosomal dominant hypocalcemia 1

10.1101/2021.02.01.429170 ◽

2021 ◽

Author(s):

Wouter H. van Megen ◽

Rebecca Tan ◽

R. Todd Alexander ◽

Henrik Dimke

Keyword(s):

Autosomal Dominant ◽

Urinary Calcium ◽

The Body ◽

Hek293 Cells ◽

Calcium Handling ◽

Tissue Specific ◽

Symptomatic Hypocalcemia ◽

Renal Phenotype ◽

Autosomal Dominant Hypocalcemia

AbstractBackgroundParathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of CaSRs in kidneys and intestine attenuates local transepithelial Ca2+ transport. In patients with autosomal dominant hypocalcemia 1 (ADH1) due to activating CASR mutations, treatment of symptomatic hypocalcemia can be complicated by treatment-induced hypercalciuria, resulting in nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body are activated by increased extracellular Ca2+ concentrations, it is not understood why some ADH1 patients have reduced PTH, but not hypercalciuria at presentation, despite CaSR expression in both kidney and parathyroid.MethodsActivation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells.ResultsMice with a gain-of-function mutation in Casr are hypocalcemic with reduced plasma PTH levels. However, renal CaSRs are not activated as indicated by normal urinary calcium handling and unaltered renal Cldn14 expression. Activation of renal CaSRs only occurred after increasing plasma Ca2+ levels. Similarly, calcimimetic administration to wildtype mice induced hypocalcemia without activating renal CaSRs. Moreover, significant hypercalciuria was not observed in an ADH1 patient until blood Ca2+ was normalized. In vitro experiments suggest that increased CaSR levels in the parathyroid relative to the kidney contribute tissue-specific CaSR activation thresholds.ConclusionHere we delineate tissue-specific CaSR activation thresholds, where parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even with overactivating mutations. These findings may aid in the management of ADH1 patients.

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Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

Scientific Reports ◽

10.1038/s41598-021-90039-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

George Khelashvili ◽

Anoop Narayana Pillai ◽

Joon Lee ◽

Kalpana Pandey ◽

Alexander M. Payne ◽

...

Keyword(s):

Molecular Dynamics ◽

Retinitis Pigmentosa ◽

Molecular Dynamics Simulations ◽

Autosomal Dominant ◽

Hek293 Cells ◽

Transmembrane Helices ◽

Wild Type ◽

Dynamics Simulations ◽

G Protein Coupled

AbstractMutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

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Phenotypic Variation in FAM83H-associated Amelogenesis Imperfecta

Journal of Dental Research ◽

10.1177/0022034509333822 ◽

2009 ◽

Vol 88 (4) ◽

pp. 356-360 ◽

Cited By ~ 42

Author(s):

J.T. Wright ◽

S. Frazier-Bowers ◽

D. Simmons ◽

K. Alexander ◽

P. Crawford ◽

...

Keyword(s):

Amino Acids ◽

Mineral Content ◽

Autosomal Dominant ◽

Mutational Analysis ◽

Gene Mutations ◽

Amelogenesis Imperfecta ◽

Nonsense Mutations ◽

Deletion Mutations ◽

Biochemical Studies ◽

Normal Thickness

FAM83H gene mutations are associated with autosomal-dominant hypocalcified amelogenesis imperfecta (ADHCAI), which is typically characterized by enamel having normal thickness and a markedly decreased mineral content. This study tested the hypothesis that there are phenotype and genotype associations in families with FAM83H-associated ADHCAI. Seven families segregating ADHCAI (147 individuals) were evaluated. Phenotyping included clinical, radiographic, histological, and biochemical studies, and genotyping was by mutational analysis. Multiple novel FAM83H mutations were identified, including two 2-bp-deletion mutations, the first non-nonsense mutations identified. Craniofacial deviation from normal was more prevalent in the affected individuals. Affected individuals having truncating FAMH3H mutations of 677 or fewer amino acids presented a generalized ADHCAI phenotype, while those having mutations capable of producing a protein of at least 694 amino acids had a unique and previously unreported phenotype affecting primarily the cervical enamel. This investigation shows that unique phenotypes are associated with specific FAM83H mutations.

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Isolation and characterization of cDNA clones for rat ribophorin I: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes

The Journal of Cell Biology ◽

10.1083/jcb.104.4.855 ◽

1987 ◽

Vol 104 (4) ◽

pp. 855-863 ◽

Cited By ~ 28

Author(s):

V Harnik-Ort ◽

K Prakash ◽

E Marcantonio ◽

DR Colman ◽

MG Rosenfeld ◽

...

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Amino Acid ◽

Amino Acid Sequence ◽

Cdna Library ◽

Cdna Clone ◽

In Vitro Synthesis ◽

Coding Sequence ◽

Amino Terminal

Ribophorins I and II are two transmembrane glycoproteins that are characteristic of the rough endoplasmic reticulum and are thought to be part of the apparatus that affects the co-translational translocation of polypeptides synthesized on membrane-bound polysomes. A ribophorin I cDNA clone containing a 0.6-kb insert was isolated from a rat liver lambda gtll cDNA library by immunoscreening with specific antibodies. This cDNA was used to isolate a clone (2.3 kb) from a rat brain lambda gtll cDNA library that contains the entire ribophorin I coding sequence. SP6 RNA transcripts of the insert in this clone directed the in vitro synthesis of a polypeptide of the expected size that was immunoprecipitated with anti-ribophorin I antibodies. When synthesized in the presence of microsomes, this polypeptide, like the translation product of the natural ribophorin I mRNA, underwent membrane insertion, signal cleavage, and co-translational glycosylation. The complete amino acid sequence of the polypeptide encoded in the cDNA insert was derived from the nucleotide sequence and found to contain a segment that corresponds to a partial amino terminal sequence of ribophorin I that was obtained by Edman degradation. This confirmed the identity of the cDNA clone and established that ribophorin I contains 583 amino acids and is synthesized with a cleavable amino terminal insertion signal of 22 residues. Analysis of the amino acid sequence of ribophorin I suggested that the polypeptide has a simple transmembrane disposition with a rather hydrophilic carboxy terminal segment of 150 amino acids exposed on the cytoplasmic face of the membrane, and a luminal domain of 414 amino acids containing three potential N-glycosylation sites. Hybridization measurements using the cloned cDNA as a probe showed that ribophorin I mRNA levels increase fourfold 15 h after partial hepatectomy, in confirmation of measurements made by in vitro translation of liver mRNA. Southern blot analysis of rat genomic DNA suggests that there is a single copy of the ribophorin I gene in the haploid rat genome.

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INTRACELLULAR SYNTHESIS, TRANSPORT, AND PACKAGING OF PROTEINACEOUS YOLK IN OOCYTES OF ORCONECTES IMMUNIS

The Journal of Cell Biology ◽

10.1083/jcb.52.2.420 ◽

1972 ◽

Vol 52 (2) ◽

pp. 420-437 ◽

Cited By ~ 47

Author(s):

L. R. Ganion ◽

R. G. Kessel

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Rna Synthesis ◽

Actinomycin D ◽

Maternal Blood ◽

Blood Proteins ◽

Yolk Granules ◽

Early Phases

The incorporation of leucine-3H into either ovarian or oocyte proteins occurs throughout vitellogenesis, but is at a maximum during early phases of this process. The labeling of ovarian and oocyte proteins is inhibited with cycloheximide. Oocytes are permeable to actinomycin D, and this drug does not affect the incorporation of amino acids into oocyte proteins but does block oocyte RNA synthesis. By means of both light microscope and high resolution radioautography, it has been demonstrated that the initial incorporation of leucine-3H under both in vitro and in vivo conditions occurs in elements of the rough-surfaced endoplasmic reticulum in the oocyte. Under pulse-chase conditions, the label subsequently becomes associated with intracisternal (precursor yolk) granules now aggregated within the cisternae of the connected smooth-surfaced endoplasmic reticulum. By 7 days, mature yolk globules are extensively labeled. The results of experiments designed to assess the possible contribution of maternal blood proteins to yolk deposition indicate that such a contribution is minimal. It is concluded that the crayfish oocyte is programmed for and capable of synthesizing the massive store of proteinaceous yolk present in the egg at the end of oogenesis.

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