scholarly journals Genetically encoded multimode reporter of adaptor protein 3 (AP-3) traffic in budding yeast

2018 ◽  
Author(s):  
Rachael L. Plemel ◽  
Greg Odorizzi ◽  
Alexey J. Merz
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Budding Yeast ◽  
Agar Plate ◽  
Adaptor Protein ◽  
Epileptic Encephalopathy ◽  
Early Endosomes ◽  
Hermansky Pudlak Syndrome ◽  
Functional Deficiency

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening.SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathway’s importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

scholarly journals AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

2001 ◽  
Vol 12 (7) ◽  
pp. 2075-2085 ◽  
Author(s):  
Marjan Huizing ◽  
Rangaprasad Sarangarajan ◽  
Erin Strovel ◽  
Yang Zhao ◽  
William A. Gahl ◽  
...  
Keyword(s):  
Dense Body ◽  
Multivesicular Bodies ◽  
Normal Pattern ◽  
Late Endosomes ◽  
Human Melanocytes ◽  
Hermansky Pudlak Syndrome ◽  
Functional Deficiency ◽  
Tyrosinase Related Protein

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the β3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the β3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3–deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

scholarly journals A dengue type 2 reporter virus assay amenable to high-throughput screening

2020 ◽  
Vol 183 ◽  
pp. 104929
Author(s):  
Li-Hsin Li ◽  
Suzanne J.F. Kaptein ◽  
Michael A. Schmid ◽  
Joanna Zmurko ◽  
Pieter Leyssen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Reporter Virus ◽  
Virus Assay

scholarly journals High-glucose 3D INS-1 cell model combined with a microfluidic circular concentration gradient generator for high throughput screening of drugs against type 2 diabetes

RSC Advances ◽  
2018 ◽  
Vol 8 (45) ◽  
pp. 25409-25416 ◽  
Author(s):  
Yong Luo ◽  
Xiuli Zhang ◽  
Yujiao Li ◽  
Jiu Deng ◽  
Xiaorui Li ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Pharmaceutical Industry ◽  
High Throughput ◽  
Concentration Gradient ◽  
High Glucose ◽  
High Throughput Screening ◽  
Cell Model ◽  
Gradient Generator

In vitro models for screening of drugs against type 2 diabetes are crucial for the pharmaceutical industry.

scholarly journals Recent Advances in In-Vitro Assays for Type 2 Diabetes Mellitus: An Overview

2020 ◽  
Vol 16 (1) ◽  
pp. 13-23
Author(s):  
Nazmina Vhora ◽  
Ujjal Naskar ◽  
Aishwarya Hiray ◽  
Abhijeet S. Kate ◽  
Alok Jain
Keyword(s):  
Type 2 Diabetes ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antidiabetic Activity ◽  
Screening Methods ◽  
Selection Of

BACKGROUND: A higher rate of attenuation of molecules in drug discovery has enabled pharmaceutical companies to enhance the efficiency of their hit identification and lead optimization. Selection and development of appropriate in-vitro and in-vivo strategies may improve this process as primary and secondary screening utilize both strategies. In-vivo approaches are too relentless and expensive for assessing hits. Therefore, it has become indispensable to develop and implement suitable in-vitro screening methods to execute the required activities and meet the respective targets. However, the selection of an appropriate in-vitro assay for specific evaluation of cellular activity is no trivial task. It requires thorough investigation of the various parameters involved. AIM: In this review, we aim to discuss in-vitro assays for type 2 diabetes (T2D), which have been utilized extensively by researchers over the last five years, including target-based, non-target based, low-throughput, and high-throughput screening assays. METHODS: The literature search was conducted using databases including Scifinder, PubMed, ScienceDirect, and Google Scholar to find the significant published articles. DISCUSSION and CONCLUSION: The accuracy and relevance of in-vitro assays have a significant impact on the drug discovery process for T2D, especially in assessing the antidiabetic activity of compounds and identifying the site of effect in high-throughput screening. The report reviews the advantages, limitations, quality parameters, and applications of the probed invitro assays, and compares them with one another to enable the selection of the optimal method for any purpose. The information on these assays will accelerate numerous procedures in the drug development process with consistent quality and accuracy.

scholarly journals Quantitative, High-Throughput Assays for Proteolytic Degradation of Amylin

2020 ◽  
Vol 3 (4) ◽  
pp. 81
Author(s):  
Caitlin N. Suire ◽  
Monica K. Brizuela ◽  
Malcolm A. Leissring
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Peptide Hormone ◽  
Proteolytic Degradation ◽  
Insulin Degrading Enzyme ◽  
Islet Amyloid ◽  
Health And Disease ◽  
Pancreatic Peptide

Amylin is a pancreatic peptide hormone that regulates glucose homeostasis but also aggregates to form islet amyloid in type-2 diabetes. Given its role in both health and disease, there is renewed interest in proteolytic degradation of amylin by insulin-degrading enzyme (IDE) and other proteases. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. The FrAB-based substrate can be used in a readily implemented fluorescence-based protocol or in a fluorescence polarization (FP)-based protocol that is more amenable to high-throughput screening (HTS), whereas the FRAM substrate has the advantage of permitting continuous monitoring of proteolytic activity. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications.

High-throughput screening of food additives with synergistic effects on high hydrostatic pressure inactivation of budding yeast

2019 ◽  
Vol 39 (2) ◽  
pp. 280-292
Author(s):  
Akinori Iguchi ◽  
Masaki Ikarashi ◽  
Akiko Maruyama ◽  
Saori (Ujiie) Hori ◽  
Kazuki Nomura ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
High Throughput ◽  
High Hydrostatic Pressure ◽  
High Throughput Screening ◽  
Budding Yeast ◽  
Synergistic Effects ◽  
Food Additives

scholarly journals Biosensor-based multi-gene pathway optimization for enhancing the production of glycolate

2021 ◽  
Author(s):  
Shumin Xu ◽  
Linpei Zhang ◽  
Shenghu Zhou ◽  
Yu Deng
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Metabolic Flux ◽  
Agar Plate ◽  
Expression Level ◽  
Rapid Screening ◽  
Screening Methods ◽  
Synthetic Pathway ◽  
Gene Pathway ◽  
Pathway Optimization

Glycolate is widely used in industry, especially in the fields of chemical cleaning, cosmetics, and medical materials, and has broad market prospects for the future. Recent advances in metabolic engineering and synthetic biology have significantly improved the titer and yield of glycolate. However, an expensive inducer was used in previous studies that is not feasible for use in large-scale industrial fermentations. To constitutively biosynthesize glycolate, the expression level of each gene of the glycolate synthetic pathway needs to be systemically optimized. The main challenge of multi-gene pathway optimization is being able to select or screen the optimum strain from the randomly assembled library by an efficient high-throughput method within a short period of time. To overcome these challenges, we firstly established a glycolate-responsive biosensor and developed agar plate- and 48-well deep well plate-scale high-throughput screening methods for rapid screening of superior glycolate producers from a large library. A total of 22 gradient strength promoter-5′-UTR complexes were randomly cloned upstream of the genes of the glycolate synthetic pathway, generating a large random assembled library. After rounds of screening, the optimum strain was obtained from 6×105 transformants in a week, and it achieved a titer of 40.9 ± 3.7 g/L glycolate in a 5-L bioreactor. Furthermore, high expression levels of the enzymes YcdW and GltA were found to promote glycolate production, whereas AceA has no obvious impact on glycolate production. Overall, the glycolate biosensor-based pathway optimization strategy presented in this work provides a paradigm for other multi-gene pathway optimizations. Importance The use of strong promoters, such as pTrc and T7, to control gene expression not only need adding expensive inducers but also results in excessive protein expression that may be resulting in unbalanced metabolic flux and the waste of cellular building blocks and energy. To balance the metabolic flux of glycolate biosynthesis, the expression level of each gene needs to be systemically optimized in a constitutive manner. However, the lack of a high-throughput screening methods restricted the glycolate synthetic pathway optimization. Our work firstly established a glycolate-response biosensor, then agar plate and 48-well plate scale high-throughput screening methods were developed for rapid screening of optimum pathways from a large library. Finally, we obtained a glycolate producing strain with good biosynthetic performance, and the use of the expensive inducer IPTG was avoided, which broadens our understanding about the mechanism of glycolate synthesis.

scholarly journals BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

2007 ◽  
Vol 18 (3) ◽  
pp. 768-780 ◽  
Author(s):  
Subba Rao Gangi Setty ◽  
Danièle Tenza ◽  
Steven T. Truschel ◽  
Evelyn Chou ◽  
Elena V. Sviderskaya ◽  
...  
Keyword(s):  
Protein Complex ◽  
Genetic Disorder ◽  
Adaptor Protein ◽  
Transport Pathways ◽  
Cargo Transport ◽  
Early Endosomes ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
And Function ◽  
Tyrosinase Related Protein

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1–deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.

scholarly journals Interstitial Lung Disease and Pulmonary Fibrosis in Hermansky-Pudlak Syndrome Type 2, an Adaptor Protein-3 Complex Disease

2011 ◽  
Vol 18 (1) ◽  
pp. 56-64 ◽  
Author(s):  
Bernadette R. Gochuico ◽  
Marjan Huizing ◽  
Gretchen A. Golas ◽  
Charles D. Scher ◽  
Maria Tsokos ◽  
...  
Keyword(s):  
Interstitial Lung Disease ◽  
Pulmonary Fibrosis ◽  
Lung Disease ◽  
Complex Disease ◽  
Adaptor Protein ◽  
Syndrome Type ◽  
Hermansky Pudlak Syndrome

scholarly journals High-throughput screening of high protein producer budding yeast using gel microdrop technology

2019 ◽  
Author(s):  
Hirotsugu Fujitani ◽  
Soichiro Tsuda ◽  
Tomoko Ishii ◽  
Masayuki Machida
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Production ◽  
Budding Yeast ◽  
Screening Method ◽  
Screening Strategy ◽  
Secreted Proteins ◽  
Cell Sorter ◽  
Emulsion Method ◽  
Mutagenized Cell

AbstractThe need for protein production has been growing over the years in various industries. We here present a high-throughput screening strategy to isolate high producer budding yeast clones from a mutagenized cell population using gel microdrop (GMD) technology. We use a microfluidic water-in-oil (W/O) emulsion method to produce monodisperse GMDs and a microfluidic cell sorter for damage-free sorting of GMDs by fluorescently quantifying secreted proteins. As a result, this high-throughput GMD screening method effectively selects high producer clones and improves protein production up to five-fold. We speculate that this screening strategy can be applied, in principle, to select any types of high producer cells (bacterial, fungal, mammalian, etc.) which produce arbitrary target protein as it does not depend on enzymes to be produced.

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