A genetic bistable switch utilizing nonlinear protein degradation

Background

Fluorescent reporter proteins have revolutionized our understanding of cellular bioprocesses by enabling live cell imaging with exquisite spatio-temporal resolution. Existing fluorescent proteins are predominantly based on the green fluorescent protein (GFP) and related analogs. However, GFP-family proteins strictly require molecular oxygen for maturation of fluorescence, which precludes their application for investigating biological processes in low-oxygen environments. A new class of oxygen-independent fluorescent reporter proteins was recently reported based on flavin-binding photosensors from Bacillus subtilis and Pseudomonas putida. However, flavin-binding fluorescent proteins show very limited brightness, which restricts their utility as biological imaging probes.

Results

In this work, we report the discovery of bright mutants of a flavin-binding fluorescent protein from P. putida using directed evolution by site saturation mutagenesis. We discovered two mutations at a chromophore-proximal amino acid (F37S and F37T) that confer a twofold enhancement in brightness relative to the wild type fluorescent protein through improvements in quantum yield and holoprotein fraction. In addition, we observed that substitution with other aromatic amino acids at this residue (F37Y and F37W) severely diminishes fluorescence emission. Therefore, we identify F37 as a key amino acid residue in determining fluorescence.

Conclusions

To increase the scope and utility of flavin-binding fluorescent proteins as practical fluorescent reporters, there is a strong need for improved variants of the wild type protein. Our work reports on the application of site saturation mutagenesis to isolate brighter variants of a flavin-binding fluorescent protein, which is a first-of-its-kind approach. Overall, we anticipate that the improved variants will find pervasive use as fluorescent reporters for biological studies in low-oxygen environments.     

Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.     

A human phenome-interactome network of protein complexes implicated in genetic disorders

We performed a systematic, large-scale analysis of human protein complexes comprising gene products implicated in many different categories of human disease to create a phenome-interactome network. This was done by integrating quality-controlled interactions of human proteins with a validated, computationally derived phenotype similarity score, permitting identification of previously unknown complexes likely to be associated with disease. Using a phenomic ranking of protein complexes linked to human disease, we developed a Bayesian predictor that in 298 of 669 linkage intervals correctly ranks the known disease-causing protein as the top candidate, and in 870 intervals with no identified disease-causing gene, provides novel candidates implicated in disorders such as retinitis pigmentosa, epithelial ovarian cancer, inflammatory bowel disease, amyotrophic lateral sclerosis, Alzheimer disease, type 2 diabetes and coronary heart disease. Our publicly available draft of protein complexes associated with pathology comprises 506 complexes, which reveal functional relationships between disease-promoting genes that will inform future experimentation.     

Defective protein kinase C-mediated actions in cystic fibrosis neutrophils

Neutrophils from cystic fibrosis (CF) patients have been shown previously to be defective in their response (beta-glucuronidase exocytosis, NADPH oxidase activation) to the chemotactic peptide FMLP. In this work, we attempted to identify the defective step in this response. We showed that stimulated CF and control neutrophils do not differ in the formation of inositol phosphates. On the other hand, direct stimulation of protein kinase C with phorbol myristate acetate (PMA) revealed a subnormal stimulation of beta-glucuronidase exocytosis in CF neutrophils. Furthermore, retroinhibition exerted by PMA-activated protein kinase C on stimulated inositol phosphates or on beta-glucuronidase exocytosis was marginal or absent in CF neutrophils, whereas it was significant in the case of control neutrophils. Our observations suggest that the CFTR gene is expressed in neutrophils and is involved in protein kinase C-mediated actions.     

Defective protein phosphorylation in renal medulla of vasopressin-resistant mice

Protein phosphorylation was compared in renal medulla of normal mice and a strain with hereditary vasopressin-resistant diabetes insipidus (DI mice). The phosphorylation of a specific pair of proteins (M_r 185,000 and 175,000) in the particulate fraction was found to be impaired in DI mice in a fashion that correlated with the severity of the concentrating defect. Defective protein phosphorylation in DI mice may be related to the pathogenesis of cellular unresponsiveness to vasopressin.     

Evaluating genetic heterogeneity in complex disorders

OBJECTIVES: The Admixture test is routinely used in linkage analysis to take account of genetic heterogeneity, and yields an estimate of the proportion of families (alpha) segregating the linked disease gene. In complex disorders, the assumptions of the Admixture test are violated. We therefore explore how the estimate of alpha relates to the true proportion of linked families with a complex disorder in a population or dataset. METHODS: We simulated a two-locus heterogeneity model and varied genetic parameters, ascertainment scheme and phenocopy frequency. RESULTS: In this model, alpha is almost always overestimated, by as little as 5% to as much as 60%. The bias is largely attributable to (1). intrafamilial heterogeneity arising from ascertainment of families with many affected members or from analysis of dense pedigrees; (2). low informativeness, which occurs in the presence of reduced penetrance; and (3). differences in the evidence for linkage in linked and unlinked families. This bias is also affected by the analysis phenocopy frequency, but only if the linked locus is dominant and the unlinked locus is recessive. CONCLUSIONS: We conclude that, in complex diseases, the Admixture test has greater value in detecting linkage than in estimating the proportion of linked families in a dataset.     

Targeted protein degradation

The ubiquitin-proteasome pathway plays a major role in cellular protein destruction and regulates fundamental cellular processes such as the cell cycle, cell signaling, and development. By altering the substrate recognition of ubiquitin-protein ligases, their robust proteolytic activity can be re-directed to recruit and accelerate the degradation of other cellular targets. Two approaches have been applied for targeted proteolysis: one entails designing a chimeric substrate receptor for recruitment of the target protein, the other involves the construction of peptide-small-molecule hybrids that bridge the interaction between the intended target and the substrate receptor of the known ubiquitin-protein ligases. The engineered ubiquitin-proteolytic apparatus operates at the post-translational level, and thus provides a new tool of reverse genetics to dissect complicated protein functions at a higher resolution than knockout or knockdown approaches functioning at the level of DNA or RNA. It also sheds light on novel therapeutic strategies for the amelioration of human disease.     

Prostacyclin degradation in patients with quantitative platelet disorders

Plasma prostacyclin (PGI2) degradation rates were measured at 1, 5, 15 and 30 min in a group of patients with platelet quantitative disorders of various pathogeneses, including 13 with thrombocytosis, 16 with thrombocytopenia from impaired production in the bone marrow, 11 with thrombocytopenia from peripheral destruction, and 28 normal, healthy persons. Patients with thrombocytosis had a low PGI2 degradation rate, whereas patients with thrombocytopenia due to impaired production had a high PGI2 degradation rate. Of the patients with thrombocytopenia caused by peripheral destruction, six with idiopathic thrombocytopenia purpura (ITP) had a slow PGI2 degradation in contrast to five with systemic lupus erythematosus (SLE) - four concurrently had cryoglobulinemia - who had a rapid PGI2 degradation. The findings suggest that: (1) a platelet-derived substance in the human plasma may have a PGI2 stabilising activity; (2) presence of cryoglobulin or immune complex in plasma may interfere with PGI2 stability.     

Genomic imprinting and genetic disorders in man

In a considerable number of genetic disorders in the human, the phenotypic expression of the disease can depend on maternal or paternal inheritance of the mutation. It is suggested that genomic imprinting, an epigenetic process that marks maternal and paternal chromosomes in mammals, is involved in such parental effects.     

Lysosomes and protein degradation

Evidence from studies on mouse peritoneal macrophages using the inhibitor pepstatin confirms lysosomal involvement in basal protein degradation, and extends its relevance to degradation of long half-life and analogue containing proteins. Studies on the ability of MRC-5 (a limited life-span fibroblast line) cells to selectively degrade analogue-containing proteins are described. These indicate that this capacity is retained even in very old cells; indeed such cells show an increased proportion of rapidly-degradable proteins. Analogue containing proteins bind preferentially to lysosomal membranes, and like liver cytosol proteins of short half-life, are selectively endocytosed and degraded by certain cells in culture. Thus membrane binding allowing selective entry to the lysosomal system may be important in controlling rate of degradation of both intracellular and extracellular protein. A method potentially allowing for determination of the rate of autophagy in cells, is described. This should enable further assessment of the quantitative involvement of lysosomes in protein degradation.