Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator

Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.     

From membrane to molecule to the third amino acid from the left with a membrane transport protein

The lac permease of E. coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer. The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane. Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover. Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E. coli is proposed. Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII). In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles. Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa. Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa. In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex. In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325. The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane. Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism. On the other hand, widespread, cooperative conformational changes appear to be involved in turnover. As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover. This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e. helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism. (ABSTRACT TRUNCATED)     

Spectrum of mutations in the OCRL1 gene in the Lowe oculocerebrorenal syndrome

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder characterized by congenital cataracts, mental retardation, and renal Fanconi syndrome. The OCRL1 gene, which, when mutated, is responsible for OCRL, encodes a 105-kD Golgi protein with phosphatidylinositol (4,5)bisphosphate (PtdIn[4,5]P2) 5-phosphatase activity. We have examined the OCRL1 gene in 12 independent patients with OCRL and have found 11 different mutations. Six were nonsense mutations, and one a deletion of one or two nucleotides that leads to frameshift and premature termination. In one, a 1.2-kb genomic deletion of exon 14 was identified. In four others, missense mutations or the deletion of a single codon were found to involve amino acid residues known to be highly conserved among proteins with PtdIns(4,5)P2 5-phosphatase activity. All patients had markedly reduced PtdIns(4,5)P2 5-phosphatase activity in their fibroblasts, whereas the ocrl1 protein was detectable by immunoblotting in some patients with either missense mutations or a codon deletion but was not detectable in those with premature termination mutations. These results confirm and extend our previous observation that the OCRL phenotype results from loss of function of the ocrl1 protein and that mutations are generally heterogeneous. Missense mutations that abolish enzyme activity but not expression of the protein will be useful for studying structure-function relationships in PtdIns(4,5)P2 5-phosphatases.     

Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.     

Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coli lie in close proximity [Jung, K., Jung, H., Wu, J., Privé, G.G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. In this study, Glu269 was replaced with His in wild-type permease, leading to the presence of bis-His residues between helices VIII and X. Wild-type and Glu269-->His permease containing a biotin acceptor domain were purified by monomeric avidin affinity chromatography, and binding of Mn2+ was studied by electron paramagnetic resonance (EPR) spectroscopy. The amplitude of the Mn2+ EPR spectrum is reduced by the Glu269-->His mutant, while no change is observed in the presence of wild-type permease. The Glu269-->His mutant contains a single binding site for Mn2+ with a KD of about 43 microM, and Mn2+ binding is pH dependent with no binding at pH 5.0, stoichiometric binding at pH 7.5, and a midpoint at about pH 6.3. The results confirm the conclusion that helices VIII and X are closely opposed in the tertiary structure of lac permease and provide a novel approach for studying helix proximity, as well as solvent accessibility, in polytopic membrane proteins.     

Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer

BACKGROUND & AIMS: Germline mutations in four DNA mismatch repair genes are known to cause susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). The rapidly increasing information about these mutations needs to be collected and appropriately stored to facilitate further studies on the biological and clinical significance of the findings. METHODS: The International Collaborative Group on HNPCC has established a database of DNA mismatch repair gene mutations and polymorphisms. In this report, 126 predisposing mutations were analyzed. RESULTS: A majority of the mutations affected either the Mut L homologue (MLH) 1 (n = 75) or the Mut S homologue (MSH) 2 (n = 48) and were quite evenly distributed, with some clustering in MSH2 exon 12 and MLH1 exon 16. Most MSH2 mutations consisted of frameshift (60%) or nonsense changes (23%), whereas MLH1 was mainly affected by frameshift (40%) or missense alterations (31%). Although most mutations were unique, a few common recurring mutations were identified. Of the families studied (n = 202), 82% met the Amsterdam criteria and 15% did not; the general mutation profile was similar in both groups. CONCLUSIONS: The construction of mutation profiles will facilitate the development of diagnostic strategies in HNPCC.     

Missense mutations in the PAX6 gene in aniridia

PURPOSE: Aniridia is caused by a mutation of the PAX6 gene. Haploinsufficiency of the gene product is thought to result in the aniridia phenotype, because most mutations thus far detected have been large deletions encompassing the entire gene and nonsense, frameshift, or splice errors that result in premature translational termination on one of the alleles. Only two missense mutations have been detected in aniridia pedigrees, each of which occurs in its paired domain or homeodomain. In this study, four novel missense mutations were found in three aniridia pedigrees. METHODS: Polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing of the PAX6 gene were performed using genomic DNA of three aniridia pedigrees and more than 100 healthy control subjects. RESULTS: Three mutations occurred in the N-terminal subdomain of the paired domain, namely N17S, I29V, and R44Q, the first two of which were detected on the same allele of one patient. The other mutation (Q178H) was in the linking portion of the paired domain and homeodomain. CONCLUSIONS: These missense mutations give rise to haploinsufficiency by another route, because the missense mutations presented here resulted in an aniridia phenotype indistinguishable from that caused by a heterozygous deletion of the entire PAX6 gene.     

The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase

Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR). The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTP-binding protein Ran. In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDEdelta) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR392). The interaction of RPGR with PDEdelta was confirmed by pull-down assays and plasmon surface resonance. The binding affinity was determined to be 90 nM. Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system. All missense mutations showed reduced interaction with PDEdelta. A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDEdelta. PDEdelta is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family. These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.     

Mutations of conserved cysteine residues in the CWLC motif of the oncoretrovirus SU protein affect maturation and translocation

The envelope glycoprotein complex is composed of two polypeptides, an external heavily glycosylated polypeptide (SU) and a membrane-spanning protein (TM). Together they form a heterodimer on the surface of the virion. These proteins are synthesized in the form of a polyprotein precursor which is glycosylated and proteolytically processed during its maturation in the secretory pathway. A highly conserved stretch of four amino acids, CWLC, has been identified in most known oncoretroviral SU proteins, about two-thirds of the distance from the amino terminus. To study the significance of this sequence for the structure and/or function of SU, cysteine to serine mutations were made in reticuloendotheliosis virus strain A. Initial studies showed that substitution of either one or both cysteines resulted in the production of noninfectious virus. Furthermore, immunoprecipitations and pulse-chase analysis demonstrated that the mutants yielded envelope polyprotein precursors which were stable. However, the polyprotein precursors were not proteolytically processed into SU and TM, and immunoprecipitations indicate that the immature polyproteins form aggregates, suggesting that the mutations interfere with proper folding. Although not proteolytically processed, at least one of the mutant glycoproteins appeared to be efficiently transported to the cell surface. These studies indicate that changing either cysteine residue abrogates viral infectivity by affecting folding, inhibiting normal maturation of the envelope glycoproteins.     

Genetically probing the regions of ribose-binding protein involved in permease interaction

The ribose-binding protein (RBP) of Escherichia coli, located in the periplasm, binds to ribose and mediates transport and chemotaxis. The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz. Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene. The expression of ompC can be increased by an interaction of ribose-bound RBP with Trz. A transport defect, either in the binding protein or in the membrane permease, causes a signalling-constitutive Lac+ phenotype of Trz even in the absence of ribose. This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high-affinity transport system. By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction. Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar-binding properties were not substantially affected. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor.     

Folding of the presequence of yeast pAPI into an amphipathic helix determines transport of the protein from the cytosol to the vacuole

To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport. 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix. The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix. Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent. Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport. Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen. Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen. Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair. The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4. Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport. These results provide direct evidence of the involvement of the presequence in the transport of pAPI from the cytosol to the vacuole. They show that folding of the pAPI presequence is conditioned by the physical/chemical properties of the environment and is critical for targeting the protein to the vacuolar membrane and for its translocation to the vacuolar lumen.     

Opposing roles of the holliday junction processing systems of Escherichia coli in recombination-dependent adaptive mutation

Aspects of the molecular mechanism of "adaptive" mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.     

Neural activity related to reaching and grasping in rostral and caudal regions of rat motor cortex

We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase alpha-primase and DNA polymerase beta. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV-tk) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N-ethyl-N-nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G-->A transition mutations was observed for both polymerases, as expected due to mispairing by O6-ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase beta and polymerase alpha-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase alpha-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G-->A transition frequency. In the polymerase beta reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G-->A transition errors by polymerase beta. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase.     

Exon scanning for mutation of the NF2 gene in schwannomas

Family studies and tumor analyses have combined to indicate that neurofibromatosis 2 (NF2), a disorder characterized by multiple benign tumors of the nervous system, and sporadic non-inherited forms of the same tumor types are both caused by inactivation of a tumor suppressor gene located in 22q12. Recently, the gene encoding merlin, a novel member of a family of cytoskeleton-associated proteins, was identified as the NF2 tumor suppressor. To facilitate the search for merlin mutations, we have defined the exon-intron boundaries for all 17 NF2 exons, including one subject to alternative splicing. We have developed polymerase chain reaction assays to amplify each exon from genomic DNA, and used these assays to perform single-strand conformation polymorphism analysis of DNA from 30 sporadic and eight NF2-derived schwannomas, the hallmark tumor type in this disorder. Of a maximum of 60 alleles scanned, 32 showed mutations affecting expression of the merlin protein. Thirty of these mutations are predicted to lead to a truncated protein due to frameshift, creation of a stop codon, or interference with normal splicing, while two are missense mutations. Thus, inactivation of merlin is a common feature underlying both inherited and sporadic forms of schwannoma.     

Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens

Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%-2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the "5T" allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for delta F508 and none was compound heterozygous for delta F508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for delta F508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the "5T allele" was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G-->A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.     

Dissection of de novo membrane insertion activities of internal transmembrane segments of ATP-binding-cassette transporters: toward understanding topological rules for membrane assembly of polytopic membrane proteins

The membrane assembly of polytopic membrane proteins is a complicated process. Using Chinese hamster P-glycoprotein (Pgp) as a model protein, we investigated this process previously and found that Pgp expresses more than one topology. One of the variations occurs at the transmembrane (TM) domain including TM3 and TM4: TM4 inserts into membranes in an N(in)-C(out) rather than the predicted N(out)-C(in) orientation, and TM3 is in cytoplasm rather than the predicted N(in)-C(out) orientation in the membrane. It is possible that TM4 has a strong activity to initiate the N(in)-C(out) membrane insertion, leaving TM3 out of the membrane. Here, we tested this hypothesis by expressing TM3 and TM4 in isolated conditions. Our results show that TM3 of Pgp does not have de novo N(in)-C(out) membrane insertion activity whereas TM4 initiates the N(in)-C(out) membrane insertion regardless of the presence of TM3. In contrast, TM3 and TM4 of another polytopic membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), have a similar level of de novo Nin-Cout membrane insertion activity and TM4 of CFTR functions only as a stop-transfer sequence in the presence of TM3. Based on these findings, we propose that 1) the membrane insertion of TM3 and TM4 of Pgp does not follow the sequential model, which predicts that TM3 initiates N(in)-C(out) membrane insertion whereas TM4 stops the insertion event; and 2) "leaving one TM segment out of the membrane" may be an important folding mechanism for polytopic membrane proteins, and it is regulated by the N(in)-C(out) membrane insertion activities of the TM segments.     

The transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus G protein

The transmembrane (TM) domains of viral fusion proteins are required for fusion, but their precise role is unknown. G protein, the fusion protein of vesicular stomatitis virus, was previously shown to lose syncytia-forming ability if six residues (GLIIGL) were deleted from its TM domain. The 20-residue TM domain of wild-type (TM20) G protein was thus changed into a TM domain of 14 residues (TM14). To assess possible sequence specificity for this loss of function, the two Gly residues in TM20 were replaced with either Ala or Leu. Both mutations resulted in complete loss of fusion activity, as measured by fusion-dependent reporter gene transfer. Single substitutions decreased activity by about half. TM14 was weakly active (15%) but reintroduction of a Gly residue into TM14 by a single Ile --> Gly substitution increased activity to 80%. All mutants retained normal hemifusion activity, i.e., lipid mixing between the outer leaflets of the reacting membranes. Thus, at least one TM Gly residue is required for a late step in fusion mediated by G protein. Gly residues were significantly (2.6-fold; P = 0.004) more abundant in the TM domains of viral fusion proteins than in those of nonfusion proteins and were distributed differently within the TM domain. Thus, Gly residues in the TM domain of other viral fusion proteins may also prove to be important for fusion activity.     

Mutation analysis of the MEN1 gene in multiple endocrine neoplasia type 1, familial acromegaly and familial isolated hyperparathyroidism

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal dominant disease characterized by neoplasia of the parathyroid glands, the endocrine pancreas, and the anterior pituitary gland. In addition, families with isolated endocrine neoplasia, notably familial isolated hyperparathyroidism (FIHP) and familial acromegaly, have also been reported. However, whether these families constitute MEN 1 variants or separate entities remains speculative as the genetic bases for these diseases are unclear. The gene for MEN 1 has recently been cloned and characterized. Using single strand conformation analysis (SSCA) and sequencing, we performed mutation analysis in: a) a total of 55 MEN 1 families from 7 countries, b) 13 isolated MEN 1 cases without family history of the disease, c) 8 acromegaly families, and d) 4 FIHP families. Mutations were identified in 27 MEN 1 families and 9 isolated cases. The 22 different mutations spread across most of the 9 translated exons and included frameshift (11), nonsense (6), splice (2), missense mutations (2), and in-frame deletions (1). Among the 19 Finnish MEN 1 probands, a 1466del12 mutation was identified in 6 families with identical 11q13 haplotypes and in 2 isolated cases indicating a common founder. One frameshift mutation caused by 359del4 (GTCT) was found in 1 isolated case and 4 kindreds of different origin and haplotypes; this mutation therefore represents a common "warm" spot in the MEN1 gene. By analyzing the DNA of the parents of an isolated case one mutation was confirmed to be de novo. No mutation was found in any of the acromegaly and small FIHP families, suggesting that genetic defects other than the MEN1 gene might be involved and that additional such families need to be analyzed.     

Immunosuppressant activity in human beta-casein fragment analogs

X-linked retinitis pigmentosa (XLRP) results from mutations in at least two different loci, designated RP2 and RP3, located at Xp11.3 and Xp21.1, respectively. The RP3 gene was recently isolated by positional cloning, whereas the RP2 locus was mapped genetically to a 5-cM interval. We have screened this region for genomic rearrangements by the YAC representation hybridization (YRH) technique and detected a LINE1 (L1) insertion in one XLRP patient. The L1 retrotransposition occurred in an intron of a novel gene that consisted of five exons and encoded a polypeptide of 350 amino acids. Subsequently, nonsense, missense and frameshift mutations, as well as two small deletions, were identified in six additional patients. The predicted gene product shows homology with human cofactor C, a protein involved in the ultimate step of beta-tubulin folding. Our data provide evidence that mutations in this gene, designated RP2, are responsible for progressive retinal degeneration.