Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter

Grebowski, Jacek; Studzian, Maciej; Bartosz, Grzegorz; Pułaski, Łukasz

doi:10.1016/j.bbamem.2016.06.022

Cited by 6 publications

(3 citation statements)

References 34 publications

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“…Multidrug resistance (MDR) is present in leishmania sis and as expected, ABC transporters are a major factor (Grebowski et al, 2016). Overexpression of the membrane-bound ABC transporters MDR associated protein 1 (MRP1) and P-gp (on the surfaces of leishmania s) is one of the mechanisms of antimonial resistance (Mandal et al, 2009).…”

Section: The Atp-binding Cassette (Abc)mentioning

confidence: 92%

The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy

Saleh²,

et al. 2017

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Over the years chemotherapy failure has been a vital research topic as researchers have been striving to discover reasons behind it. The extensive studies carried out on chemotherapeutic agents confirm that resistance to chemotherapy is a major reason for treatment failure. “Resistance to chemotherapy,” however, is a comprehensive phrase that refers to a variety of different mechanisms in which ATP-binding cassette (ABC) mediated efflux dominates. The ABC is one of the largest gene superfamily of transporters among both eukaryotes and prokaryotes; it represents a variety of genes that code for proteins, which perform countless functions, including drug efflux – a natural process that protects cells from foreign chemicals. Up to date, chemotherapy failure due to ABC drug efflux is an active research topic that continuously provides further evidence on multiple drug resistance (MDR), aiding scientists in tackling and overcoming this issue. This review focuses on drug resistance by ABC efflux transporters in human, viral, parasitic, fungal and bacterial cells and highlights the importance of the MDR permeability glycoprotein being the mutual ABC transporter among all studied organisms. Current developments and future directions to overcome this problem are also discussed.

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Section: The Atp-binding Cassette (Abc)mentioning

confidence: 92%

The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy

Saleh²,

et al. 2017

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“…To study the potential of ABCB6 to rescue the above described ultrastructural phenotype, we stably transfected MNT-1 cells with constructs encoding either wild-type (WT), an inactive (K629M) 17,41 or a DUH mutant (G579E) ABCB6 variant 42 Next, parental MNT-1 cells, as well as MNT-1 cells stably expressing ABCB6 variants were depleted for endogenous ABCB6 using an siRNA construct targeting a non-coding region of the gene (siABCB6#2). Western blot analysis of MNT-1 cell lysates revealed that efficient targeting of endogenous ABCB6 still allowed the overexpression of the different ABCB6 variants (Fig 3C) providing a model system for rescue experiments.…”

Section: Abcb6 Mutations Prevent the Rescue Of Normal Amyloid Fibril mentioning

confidence: 99%

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation

Bergam

Reisecker²,

Rakvács

et al. 2018

Journal of Molecular Biology

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Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.

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“…Among them, Leishmania tarentolae, a non-pathogenic parasite, has recently been investigated and employed as a potential eukaryotic expression host [8][9][10] . To date, several successful examples of using L. tarentolae as a protein expression system have been reported by our group and others [9][10][11][12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

Enhancement of Expression Level of Modified t-PA (TNKase) in Leishmania tarentolae by Induction System

Yazdi

Tofighi

Rajaee

et al. 2019

ibj

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Background:The expression of bio-therapeutic proteins in mammalian cells, such as CHO, attains high homogeneity related to post-translational modifications. Although CHO remains the most popular cell line for bestselling biotherapeutic proteins on the market, there are still drawbacks such as expensive culture media, long time line, and high drug cost. Recently, researches on a novel Leishmania protozoan system have confirmed that this low-level eukaryote could represent a competitive alternative to the mammalian cell lines. Methods: The full length of coding sequence of modified tPA TNKase (tenecteplase) was synthesized and cloned into an inducible expression vector of L. tarentolae T7-TR cells. Results: The expression of the construct was driven by a Tet-inducible promoter. A Leishmania secretory signal sequence was also added to the expression cassette to facilitate the release of the recombinant protein into the medium. The secretory recombinant protein was analyzed and confirmed by SDS-PAGE and Western blot analyses. The expression level of TNKase in this novel system of L. tarentolae was 810 IU/mL after induction, which means that the percentage of expression increases two times compared to previous models in L. tarentolae. The TNKase activity was comparable with Activase. Conclusion: Our results suggested that expressed TNK (modified tPA) is functionally compatible with Activase regarding their effect on fibrinolysis. Given the post-translational modification similarities between mammalian and L. tarentolae, it is speculated that this system is capable of producing complex proteins such as tPA similar to mammalian system, with easier manipulation and non-expensive method.

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Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter

Cited by 6 publications

References 34 publications

The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy

The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation

Enhancement of Expression Level of Modified t-PA (TNKase) in Leishmania tarentolae by Induction System

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