Graeme Wistow

Usher Syndrome 1D and Nonsyndromic Autosomal Recessive Deafness DFNB12 Are Caused by Allelic Mutations of the Novel Cadherin-Like Gene CDH23

Genes causing nonsyndromic autosomal recessive deafness (DFNB12) and deafness associated with retinitis pigmentosa and vestibular dysfunction (USH1D) were previously mapped to overlapping regions of chromosome 10q21-q22. Seven highly consanguineous families segregating nonsyndromic autosomal recessive deafness were analyzed to refine the DFNB12 locus. In a single family, a critical region was defined between D10S1694 and D10S1737, approximately 0.55 cM apart. Eighteen candidate genes in the region were sequenced. Mutations in a novel cadherin-like gene, CDH23, were found both in families with DFNB12 and in families with USH1D. Six missense mutations were found in five families with DFNB12, and two nonsense and two frameshift mutations were found in four families with USH1D. A northern blot analysis of CDH23 showed a 9.5-kb transcript expressed primarily in the retina. CDH23 is also expressed in the cochlea, as is demonstrated by polymerase chain reaction amplification from cochlear cDNA.

Lens crystallins: gene recruitment and evolutionary dynamism

In a novel evolutionary process, enzymes and stress proteins have undergone direct gene recruitment as eye lens crystallins in a number of independent events. This may have allowed a dynamic response to changing visual environment during evolution. In spite of their diversity, many crystallins may share an origin in essential developmental processes such as cell elongation.

Domain structure and evolution in α-crystallins and small heat-shock proteins

Lu-Crystallin is an evolutionarily conserved, highly stable, structural protein of the eye lens [1,2]. A region covering over 50% of the subunit amino acid sequences of both the bovine (Ycrystallin gene products, aA and tuB [3,4] has been shown to be closely similar to a region of the small heat shock proteins (hsp) of drosophila [5] and also of the nematode C~e~orha~di~js elegtwts [6]. While considerable structural detail is known for the fly-crystallins, a superfamily comprising most of the remaining protein of the lens [1,7-lo], much less is known about the structure of the subunits of the important cY-crystallin class and the related hsp although some predictions for acrystallin have been made [l l-141. Here an attempt is made to relate gene structure and internal homology to protein structure and the possible evolutionary history of these proteins, A twodomain structure is predicted for cL-crystallin and a hitherto unnoticed internal duplication is described for hsp.

Evolution of a protein superfamily: relationships between vertebrate lens crystallins and microorganism dormancy proteins.

SummaryA search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the β- and γ-crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The β- and γ-crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single γ-crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of β- and γ-crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.

Aldose reductase and ϱ-crystallin belong to the same protein superfamily as aldehyde reductase

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH‐dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40–50% homology with ϱ‐crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and ϱ‐crystallin are members of a superfamily of related proteins.

Myxococcus xanthus spore coat protein S may have a similar structure to vertebrate lens |[beta]||[gamma]|-crystallins

The Gram-negative bacterium Myxococcus xanthus has a complex life cycle1 during which large amounts of a protein of relative molecular mass (Mr) 19,000, known as protein S, are assembled into a spore surface coat by a process that specifically requires calcium ions2,3. The gene for protein S has been cloned and the DNA sequence shows that the gene product is composed of four internally repeated homologous sequences, each 40 amino acids long4. Although protein S resembles calmodulin both in its internally duplicated structure and its ability to bind calcium4, it apparently has a β-sheet secondary structure5 rather than the helix–loop–helix motifs that characterize the calmodulin family6–8. We now show that protein S has a striking homology with the β- and γ-crystallins of the vertebrate eye lens9–11 which are β-sheet proteins with internally duplicated structures. This implies that the β- and γ-crystallins evolved from already existing proteins, whose ancestors occurred in the prokaryotes. The biological function of protein S, as a closely packed, stable protein in a relatively dehydrated environment, has implications for the functions of crystalline, which are found closely packed in the lens fibre cells, where their stability is essential for maintenance of transparency.

Tandem sequence repeats in transmembrane channel proteins

The flow of ions and small molecules out of and between cells is mediated by various classes of transmembrane proteins. One group of putative channel proteins, including the abundant lens protein MIP, is widely distributed from prokaryotes to vertebrates. This article suggests that these proteins contain a structural twofold repeat and may have arisen by gene duplication. Such a model has implications for the tertiary structures of these important proteins.

Cloning, Genomic Organization, and Characterization of a Human Cholinephosphotransferase

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated usingin vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeastCPT1 gene in its interaction with SEC14 and affect cell growth.

Lens protein expression in mammals: Taxon-specificity and the recruitment of crystallins

SummaryVertebrate lenses show remarkably taxon-specific patterns of protein composition, most obviously in the recruitment of enzymes as major crystallins. Phylogenetic relationships are particularly apparent in mammals. Here we describe ν-crystallin, which is probably identical to cytosolic aldehyde dehydrogenase, lens-specifically expressed at high abundance in the elephant shrews, primitive eutherians of the family Macroscelidae, and μ-crystallin, a novel lens protein expressed in some marsupials. We have also observed that enzymes that have been recruited as crystallins in some species are also moderately abundant in the lenses of other species. This hints that the origins of enzyme-crystallins may lie in a pool of enzymes widely expressed in lenses at fairly high levels, perhaps because they have important developmental or functional roles in the tissue.

Enzyme Activity of Macrophage Migration Inhibitory Factor toward Oxidized Catecholamines

Macrophage migration inhibitory factor (MIF) is a relatively small, 12.5-kDa protein that is structurally related to some isomerases and for which multiple immune and catalytic roles have been proposed. MIF is widely expressed in tissues with particularly high levels in neural tissues. Here we show that MIF is able to catalyze the conversion of 3,4-dihydroxyphenylaminechrome and norepinephrinechrome, toxic quinone products of the neurotransmitter catecholamines 3,4-dihydroxyphenylamine and norepinephrine, to indoledihydroxy derivatives that may serve as precursors to neuromelanin. This raises the possibility that MIF participates in a detoxification pathway for catecholamine products and could therefore have a protective role in neural tissues, which as in Parkinson’s disease, may be subject to catecholamine-related cell death.