Final Report Summary - MOT GEPHYRIN (Molybdate transport in animals. Role of gephyrin)
Summary description of the project objectives
Molybdenum (Mo) is a metal essential for the majority of orgamisms; Mo takes part, in form of a molybdenum cofactor (Moco), in the active site of key enzymes that participate in crucial processes for life. Moco biosynthesis requires the uptake of Mo into the cells; organisms acquire Mo in form of the oxyanion molybdate. In animals, the genes and proteins involved in molybdate transport are unknown, and thus the process by which molybdate is acquired is not characterised. However, animal cells need Mo as an essential part of Moco biosynthesis. In mammals, Moco deficiency causes the loss of the activity of all the enzymes containing Moco (Mo-enzymes) that leads to a severe disease phenotype characterised by progressive neurological damage, resulting in early childhood death, mainly due to the deficiency of sulphite oxidase that protects the organism, in particular the brain, from elevated levels of toxic sulphite. The objectives of the project are the following:
Objective 1: Confirm and characterise the molybdate transport activity of HsMOT2 in mammalian cells
Objective 2: Study the role of Gephyrin in molybdate transport
Objective 3: Study a proposed protein interaction between Gephyrin and HsMOT2.
Description of the work performed since the beginning of the project
The overall aim of the project is to understand how animal cells transport molybdate to meet the biosynthetic need of molybdenum cofactor, and what proteins are involved in that process.
Molybdate transport measurements have been performed in cultured mammalian cells in different conditions and genetic background. For that purpose, the fellow researcher set up a molybdate transport / consumption assay for mammalian cells by adapting the protocol used for algae and yeast cells. This method was later successfully used for HEK293, COS-7 and L929 cells.
A shRNA strategy was designed and conducted in order to knockdown HsMOT2. Three different HsMOT2-shRNA constructs were made and cultured HEK293 cells were transfected using these constructs. The effect of HsMOT2-shRNA constructs were tested by measuring molybdate transport rate and HsMOT2 transcription level using semi-quantitative PCR. In parallel, also an HsMOT2 over-expression approach was performed to confirm the results obtained with the shRNA strategy.
In order to examine the subcellular localisation of HsMOT2, different constructs were created fusing HsMOT2 to GFP, mCherry or a Myc-tag. HEK293 and/or COS-7 cells transfected with these constructs were analysed by microscopy.
A novel HsMOT2 splice variant was identified and isolated. For that purpose, a rapid amplification of cDNA ends (RACE)-PCR was performed using cDNA derived from total RNA of HEK293 cells. The role of this variant in molybdate uptake / accumulation was analysed by molybdate transport measurements after over-expression in HEK293 cells.
In order to analyse the role of Gephyrin in molybdate transport, it was knocked down using an shRNA strategy. Two different Gephyrin-shRNA constructs were made and cultured HEK293 cells were transfected with these constructs. Gephyrin knockdown was tested by semi-quantitative PCR and molybdate transport rate was measured in Gephyrin-shRNA transfected cells. In addition, a double HsMOT2 and Gephyrin silencing was performed. In parallel also, a Gephyrin over-expression approach was performed to confirm the result obtained with the shRNA strategy.
A putative HsMOT2-Gephyrin interaction was analysed by means of co-localisation studies. A Gephyrin-E-domain-GFP construct was generated. This construct together with the already existing GephyrinC3variant-GFP, GephyrinC4Cvariant-GFP and MOT2-Myc-tag (also generated in this project) were used to transfect HEK293 and COS-7 cells. Transfected cells were analysed by microscopy and immunofluorescence.
Description of the main results achieved so far
The main results derived from the project are the following:
- HsMOT2 molybdate transport activity has been confirmed in mammalian cells.
- Preliminary results and in silico studies points to a plasma membrane localisation for HsMOT2 as expected for a molybdate transporter.
- HsMOT2 transcription is activated by molybdate absence, as also seen for bacterial ModABC and Arabidopsis MOT1.
- An HsMOT2 splice variant has been identified and isolated. Its molybdate transport activity has been confirmed.
- Gephyrin knockdown studies point to a link between Moco synthesis and molybdate transport.
- Preliminary co-localisation results suggest a putative interaction between Gephyrin and HsMOT2.
These results need to be confirmed complementary experiments including co-sedimentation studies.
Description of the expected final results and their potential impact and use
Overall, the main contribution of this project to the field in the confirmation of HsMOT2 as molybdate transporter in mammals and the participation of Gephyrin in the molybdate transport process. The lack of molybdate transporters has been a drag to elucidate the molybdenum uptake process in animals. Identification and characterisation of molybdate transporters and other proteins involved in this process is a first but decisive step to understand the entire molybdenum homeostasis machinery as part of the molybdenum cofactor biosynthesis pathway in animals.
Molybdenum (Mo) is a metal essential for the majority of orgamisms; Mo takes part, in form of a molybdenum cofactor (Moco), in the active site of key enzymes that participate in crucial processes for life. Moco biosynthesis requires the uptake of Mo into the cells; organisms acquire Mo in form of the oxyanion molybdate. In animals, the genes and proteins involved in molybdate transport are unknown, and thus the process by which molybdate is acquired is not characterised. However, animal cells need Mo as an essential part of Moco biosynthesis. In mammals, Moco deficiency causes the loss of the activity of all the enzymes containing Moco (Mo-enzymes) that leads to a severe disease phenotype characterised by progressive neurological damage, resulting in early childhood death, mainly due to the deficiency of sulphite oxidase that protects the organism, in particular the brain, from elevated levels of toxic sulphite. The objectives of the project are the following:
Objective 1: Confirm and characterise the molybdate transport activity of HsMOT2 in mammalian cells
Objective 2: Study the role of Gephyrin in molybdate transport
Objective 3: Study a proposed protein interaction between Gephyrin and HsMOT2.
Description of the work performed since the beginning of the project
The overall aim of the project is to understand how animal cells transport molybdate to meet the biosynthetic need of molybdenum cofactor, and what proteins are involved in that process.
Molybdate transport measurements have been performed in cultured mammalian cells in different conditions and genetic background. For that purpose, the fellow researcher set up a molybdate transport / consumption assay for mammalian cells by adapting the protocol used for algae and yeast cells. This method was later successfully used for HEK293, COS-7 and L929 cells.
A shRNA strategy was designed and conducted in order to knockdown HsMOT2. Three different HsMOT2-shRNA constructs were made and cultured HEK293 cells were transfected using these constructs. The effect of HsMOT2-shRNA constructs were tested by measuring molybdate transport rate and HsMOT2 transcription level using semi-quantitative PCR. In parallel, also an HsMOT2 over-expression approach was performed to confirm the results obtained with the shRNA strategy.
In order to examine the subcellular localisation of HsMOT2, different constructs were created fusing HsMOT2 to GFP, mCherry or a Myc-tag. HEK293 and/or COS-7 cells transfected with these constructs were analysed by microscopy.
A novel HsMOT2 splice variant was identified and isolated. For that purpose, a rapid amplification of cDNA ends (RACE)-PCR was performed using cDNA derived from total RNA of HEK293 cells. The role of this variant in molybdate uptake / accumulation was analysed by molybdate transport measurements after over-expression in HEK293 cells.
In order to analyse the role of Gephyrin in molybdate transport, it was knocked down using an shRNA strategy. Two different Gephyrin-shRNA constructs were made and cultured HEK293 cells were transfected with these constructs. Gephyrin knockdown was tested by semi-quantitative PCR and molybdate transport rate was measured in Gephyrin-shRNA transfected cells. In addition, a double HsMOT2 and Gephyrin silencing was performed. In parallel also, a Gephyrin over-expression approach was performed to confirm the result obtained with the shRNA strategy.
A putative HsMOT2-Gephyrin interaction was analysed by means of co-localisation studies. A Gephyrin-E-domain-GFP construct was generated. This construct together with the already existing GephyrinC3variant-GFP, GephyrinC4Cvariant-GFP and MOT2-Myc-tag (also generated in this project) were used to transfect HEK293 and COS-7 cells. Transfected cells were analysed by microscopy and immunofluorescence.
Description of the main results achieved so far
The main results derived from the project are the following:
- HsMOT2 molybdate transport activity has been confirmed in mammalian cells.
- Preliminary results and in silico studies points to a plasma membrane localisation for HsMOT2 as expected for a molybdate transporter.
- HsMOT2 transcription is activated by molybdate absence, as also seen for bacterial ModABC and Arabidopsis MOT1.
- An HsMOT2 splice variant has been identified and isolated. Its molybdate transport activity has been confirmed.
- Gephyrin knockdown studies point to a link between Moco synthesis and molybdate transport.
- Preliminary co-localisation results suggest a putative interaction between Gephyrin and HsMOT2.
These results need to be confirmed complementary experiments including co-sedimentation studies.
Description of the expected final results and their potential impact and use
Overall, the main contribution of this project to the field in the confirmation of HsMOT2 as molybdate transporter in mammals and the participation of Gephyrin in the molybdate transport process. The lack of molybdate transporters has been a drag to elucidate the molybdenum uptake process in animals. Identification and characterisation of molybdate transporters and other proteins involved in this process is a first but decisive step to understand the entire molybdenum homeostasis machinery as part of the molybdenum cofactor biosynthesis pathway in animals.