Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a

KChIP4a shows a high homology with other members of the family of Kv channel-interacting proteins (KChIPs) in the conserved C-terminal core region, but exhibits a unique modulation of Kv4 channel gating and surface expression. Unlike KChIP1, the KChIP4 splice variant KChIP4a has been shown to inhibit surface expression and function as a suppressor of channel inactivation of Kv4. In this study, we sought to determine whether the multitasking KChIP4a modulates Kv4 function in a clamping fashion similar to that shown by KChIP1. Injection of Kv4.3 T1 zinc mutants into Xenopus oocytes resulted in the nonfunctional expression of Kv4.3 channels. Coexpression of Kv4.3 zinc mutants with WT KChIP4a gave rise to the functional expression of Kv4.3 current. Oocyte surface labeling results confirm the correlation between functional rescue and enhanced surface expression of zinc mutant proteins. Chimeric mutations that replace the Kv4.3 N-terminus with N-terminal KChIP4a or N-terminal deletion of KChIP4a further demonstrate that the functional rescue of Kv4.3 channel tetramerization mutants depends on the KChIP4a core region, but not its N-terminus. Structure-guided mutation of two critical residues of core KChIP4a attenuated functional rescue and tetrameric assembly. Moreover, size exclusion chromatography combined with fast protein liquid chromatography showed that KChIP4a can drive zinc mutant monomers to assemble as tetramers. Taken together, our results show that KChIP4a can rescue the function of tetramerization-defective Kv4 monomers. Therefore, we propose that core KChIP4a functions to promote tetrameric assembly and enhance surface expression of Kv4 channels by a clamping action, whereas its N-terminus inhibits surface expression of Kv4 by a mechanism that remains elusive.     

Chaperones Rescue Luciferase Folding by Separating Its Domains

Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.     

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.     

Rescue of Folding Defects in ABC Transporters Using Pharmacological Chaperones

The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (ΔF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in ΔF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of ΔF508 CFTR as well as other CFTR processing mutants in the presence of 1 μM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.     

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.     

Functional Rescue of Kallmann Syndrome-associated Prokineticin Receptor 2 (PKR2) Mutants Deficient in Trafficking

Mutations in the G protein-coupled prokineticin receptor 2 (PKR2) are known to cause Kallmann syndrome and idiopathic hypogonadotropic hypogonadism manifesting with delayed puberty and infertility. Some of the mutant receptors are not routed to the cell surface; instead, they are trapped in the cellular secretory pathway. The cell-permeant agonists/antagonists have been used to rescue some membrane receptors that are not targeted onto the cell membrane. Here, we chose three disease-associated mutations (W178S, G234D, and P290S), which all resulted in retention of PKR2 intracellularly. We show that a small molecule PKR2 antagonist (A457) dramatically increased cell surface expression and rescued the function of P290S PKR2, but had no effect on W178S and G234D PKR2. Furthermore, we also tested chemical chaperone glycerol on the cell surface expression and function of PKR2 mutants. Treatment with 10% glycerol significantly increased the cell surface expression and signaling of P290S and W178S PKR2. These data demonstrate that some Kallmann syndrome-associated, intracellularly retained mutant PKR2 receptors can be functionally rescued, suggesting a potential treatment strategy for patients bearing such mutations.     

Effect of Chemical Chaperones on Glucose-Induced Lysozyme Modifications

Nonenzymatic glycation of biomacromolecules occurs due to the diabetes mellitus and ageing. A number of small molecules, known as chemical chaperones, stabilize protein conformation against thermal and chemically induced denaturation. These compounds are including: polyamines (e.g. spermine and spermidine), amino acids (e.g. lysine) and polyols (e.g. glycerol). In this study the effect of spermidine (Spd), spermine (Spm), and glycerol on glycation, structure and function of lysozyme (LZ), as an extra-cellular protein, by different techniques is investigated. LZ is incubated with or without glucose (50 or 100 mM) in the absence or presence of Spd/Spm/glycerol at 37 °C up to 16 weeks. All the observed changes of glycated-LZ in comparison with the native protein, including: increased fluorescence emission, alteration in the secondary and tertiary structure, and reduced electrophoretic mobility- indicate its structural changes that are accompanied with its reduced activity. Glucose in the presence or absence of Spd induces the protein dimerization, but glucose plus Spm induces its trimmerization. In contrast, glycerol inhibits the LZ glycation and prevents the large changes on its structure and function. Glucose binds lysine residues, decreases the protein positive charges and induces some alterations in its structure and activity. Polyamines also directly bind to LZ, increase its positive charges and hence induce more glycation; more conformational changes, oligomerization and its inactivation in the presence of glucose, but glycerol affect the protein environment and preserve protein from these harmful effects.     

Rescue of DNA Replication and Bacteriophage Production After Infection with T4 DNA Ligase Mutants

By preventing phage DNA synthesis during a critical period, conditions have been found under which DNA replication and phage production are rescued after infection with T4 DNA ligase mutants.     

Conjugation Rescue of Exocytosis Mutants in Tetrahymena thermophila Indicates the Presence of Functional Intermediates in the Regulated Secretory Pathway

ABSTRACT. Tetrahymena thermophila possesses a regulated secretory pathway in which mucin proteins are stored in dense-core granules, called mucocysts. Exocytosis-defective mutants exist that fail to secrete mucin in response to secretagogues. Four of the mutants (SB281, SB283, SB285 and SB715) appear to be blocked at different steps of the regulated secretory pathway. SB281 and SB285 accumulate mucin proteins in heterogeneous cytoplasmic organelles which have not yet been identified; SB283 makes mucocyst-like structures but they contain no immunologically identifiable 80-kDa or 50-kDa mucin proteins; and SB715 has more than normal amounts of immature and undocked mucocysts. The organelles that accumulate in exocytosis-defective mutants could be either normal intermediates in the biosynthetic pathway or aberrant structures that form as a result of the mutations. We have used conjugation rescue to analyze steps in the biogenesis of exocytosis-competent mucocysts and to identify functional intermediates. The cytoplasmic organelles that accumulate in SB281 appear to be unidentified biosynthetic intermediates, and the defect is in a cytosolic protein essential for mucocyst maturation. The organelles which accumulate in the other mutants are likely biosynthetic, but their mutations are in proteins which are labile or not free to diffuse into the mutant conjugant.     

水稻突变体与功能基因组学

突变体是功能基因组学研究的重要材料。本文综述了水稻突变体的创制方法、变异的机制、水稻突变体,基因分类和数量、克隆的水稻重要基因的研究进展,包括1698个水稻突变体,基因的分类和43个克隆的水稻突变体基因,并提出了利用水稻突变体的展望。     

Molecular Characterization and Rescue of Acatalasemic Mutants of Drosophila Melanogaster

The enzyme catalase protects aerobic organisms from oxygen-free radical damage by converting hydrogen peroxide to molecular oxygen and water before it can decompose to form the highly reactive hydroxyl radical. Hydroxyl radicals are the most deleterious of the activated oxygen intermediates found in aerobic organisms. If formed, they can react with biological molecules in their proximity; the ensuing damage has been implicated in the increasing risk of disease and death associated with aging. To study further the regulation and role of catalase we have undertaken a molecular characterization of the Drosophila catalase gene and two potentially acatalasemic alleles. We have demonstrated that a previously existing allele, Cat(n4), likely contains a null mutation, a mutation which blocks normal translation of the encoded mRNA. The Cat(n1) mutation appears to cause a significant change in the protein sequence; however, it is unclear why this change leads to a nonfunctioning protein. Viability of these acatalasemic flies can be restored by transformation with the wild-type catalase gene; hence, we conclude that the lethality of these genotypes is due solely to the lack of catalase. The availability of flies with transformed catalase genes has allowed us to address the effect of catalase levels on aging in Drosophila. Though lack of catalase activity caused decreased viability and life span, increasing catalase activity above wild-type levels had no effect on normal life span.     

Identification of an ABCB/P-glycoprotein-specific Inhibitor of Auxin Transport by Chemical Genomics

Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC₅₀ value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.     

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FH(R127H)) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-γ were able to synthesize FH(R127H), the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-γ secrete FH without retention in the ER. Moreover, the retention of FH(R127H) provoked enlargement of ER cisterns after treatment with IFN-γ. A similar ER retention was observed in Cos-7 cells expressing the mutant FH(R127H) protein. Despite this deficiency in secretion, we show that the FH(R127H) mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein.     

Functional Characterization of Oculodentodigital Dysplasia-Associated Cx43 Mutants

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

ABCB4 mediates diet-induced hypercholesterolemia in laboratory opossums

High-responding opossums are susceptible to developing hypercholesterolemia on a high-cholesterol diet, but low-responding opossums are resistant. The observation of low biliary cholesterol and low biliary phospholipids in high responders suggested that the ABCB4 gene affects response to dietary cholesterol. Two missense mutations (Arg29Gly and Ile235Leu) were found in the ABCB4 gene of high responders. High responders (ATHH strain) were bred with low responders (ATHE or ATHL strain) to produce F1 and F2 progeny in two different genetic crosses (KUSH6 and JCX) to determine the effect of ABCB4 allelic variants on plasma cholesterol concentrations after a dietary challenge. Pedigree-based genetic association analyses consistently implicated a variant in ABCB4 or a closely linked locus as a major, but not the sole, genetic contributor to variation in the plasma cholesterol response to dietary cholesterol. High responders, but not low responders, developed liver injury as indicated by elevated plasma biomarkers of liver function, probably reflecting damage to the canalicular membrane by bile salts because of impaired phospholipid secretion. Our results implicate ABCB4 as a major determinant of diet-induced hypercholesterolemia in high-responding opossums and suggest that other genes interact with ABCB4 to regulate lipemic response to dietary cholesterol.     

Functional dissection of transmembrane domains of human TAP-like (ABCB9)

An ABC transporter, TAP-Like (TAPL), was dissected into its amino-terminal transmembrane domain and the following core domain. When these domains were transiently expressed as tagged proteins with a His6- or Myc-epitope tag, the amino-terminal ones (Met¹-Lys¹⁸²) could not associate with each other, or with the full-length transporter (Met¹-Ala⁷⁶⁶). However, both the core domain (Arg¹⁴¹-Ala⁷⁶⁶) and full-length protein mutually interacted. The amino-terminal domain (Met¹-Arg¹⁴¹) as well as the full-length transporter fused with fluorescent protein GFP was sorted to lysosomal membranes upon their stable expression, as visualized by means of fluorescent microscopy, while the core domain (Arg¹⁴¹-Ala⁷⁶⁶) was broadly distributed in the intra-cellular membranes. These results suggest that the sorting signal for lysosomes is present within the amino-terminal transmembrane domain (Met¹-Arg¹⁴¹) of the TAPL molecule.