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Cloning, purification and optimization of the human mitochondrial pyruvate carriers

Lozansson, Yasmin LU (2020) KEMT30 20201
Department of Chemistry
Abstract
Diabetes is a disease that is affecting more than 422 million people worldwide. Thiazolidinedinone (TZD) an insulin sensitizer is a drug used for diabetes type 2. It was first thought that the effect of TZD was through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR- γ). Recently the mitochondrial pyruvate carrier complex (MPC) that can be found in the mitochondrial inner membrane has been found as a target for TZD. There are two subunits of MPCs in humans; MPC1 and MPC2. The two MPCs form an active complex that transports the pyruvate into the mitochondrial matrix from the cytosol. In this work, we aimed to form the human MPC complex in vitro by overproducing His-tagged MPC1 and MPC2 in the yeast Pichia... (More)
Diabetes is a disease that is affecting more than 422 million people worldwide. Thiazolidinedinone (TZD) an insulin sensitizer is a drug used for diabetes type 2. It was first thought that the effect of TZD was through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR- γ). Recently the mitochondrial pyruvate carrier complex (MPC) that can be found in the mitochondrial inner membrane has been found as a target for TZD. There are two subunits of MPCs in humans; MPC1 and MPC2. The two MPCs form an active complex that transports the pyruvate into the mitochondrial matrix from the cytosol. In this work, we aimed to form the human MPC complex in vitro by overproducing His-tagged MPC1 and MPC2 in the yeast Pichia pastoris. The proteins were solubilized with n-Dodecyl-beta-Maltoside (DDM) detergent before purified with immobilized affinity chromatography (IMAC). The IMAC fractions were analyzed on SDS-PAGE and western blot with anti-His. To form the human MPC complex MPC1 was incubated with Tobacco Etch Virus (TEV) protease to cleave off the His-tag and MPC1 was then purified with reverse IMAC. The untagged MPC1 was combined with the tagged MPC2 to form the complex and purify it with IMAC followed by SDS-PAGE and western blot with anti-His and anti-MPC1. The result from the western blot shows that in the complex both MPCs are present. This paves the way for future structural and functional studies of the human MPC complex. (Less)
Popular Abstract (Swedish)
Diabetes är en av de vanligaste folksjukdomarna runt om i världen och kan delas in i typ 1 eller typ 2 diabetes. Tiazolidindioner (TZD) är en läkemedelsgrupp som används för behandling mot diabetes typ 2 genom att öka insulinkänsligheten i skelett-, fett- och muskelceller. Det har konstaterats att den ökade insulinkänsligheten uppnås genom interaktionen med den mitokondriella pyruvattransportören (MPC). Innan MPC konstaterades som mål för TZD trodde man att effekten uppnåddes genom en interaktion med den nukleära receptorn peroxisome proliferator gamma. Genom att struktur bestämma MPC komplexet skulle en TZD analog som binder med högre affinitet till MPC och lägre affinitet till PPAR-gamma leda till mindre biverkningar. Det är av stort... (More)
Diabetes är en av de vanligaste folksjukdomarna runt om i världen och kan delas in i typ 1 eller typ 2 diabetes. Tiazolidindioner (TZD) är en läkemedelsgrupp som används för behandling mot diabetes typ 2 genom att öka insulinkänsligheten i skelett-, fett- och muskelceller. Det har konstaterats att den ökade insulinkänsligheten uppnås genom interaktionen med den mitokondriella pyruvattransportören (MPC). Innan MPC konstaterades som mål för TZD trodde man att effekten uppnåddes genom en interaktion med den nukleära receptorn peroxisome proliferator gamma. Genom att struktur bestämma MPC komplexet skulle en TZD analog som binder med högre affinitet till MPC och lägre affinitet till PPAR-gamma leda till mindre biverkningar. Det är av stort medicinskt intresse då MPC även anses vara involverad i andra sjukdomar. MPC är lokaliserat i mitokondriens inre membran och dess uppgift är att transportera pyruvat in till mitokondriematrixen. MPC komplexet består av MPC1 och MPC2 som interagerar tillsammans för att utöva deras effekt. I den här studien lyckades vi producera humant MPC1 och MPC2 var för sig i Pichia pastoris som är en jäst cell. Proteinerna renades sedan fram med affinitetskromatografi och analyserades på gel. Försök till att bilda MPC komplexet utfördes med de renade proteinerna. Resultatet från westernblot indikerar att båda MPC1 och MPC2 proteinerna finns närvarande i samma prov vilket tyder på att komplexet har bildats. Detta är ett viktigt steg mot att kunna göra detaljerade studier av hur MPC-komplexet fungerar och ser ut vilket i framtiden kan leda till nya mer effektiva läkemedel. (Less)
Please use this url to cite or link to this publication:
author
Lozansson, Yasmin LU
supervisor
organization
course
KEMT30 20201
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Protein science, Proteinvetenskap
language
English
id
9024366
date added to LUP
2020-09-14 09:36:15
date last changed
2020-09-14 09:36:15
@misc{9024366,
  abstract     = {{Diabetes is a disease that is affecting more than 422 million people worldwide. Thiazolidinedinone (TZD) an insulin sensitizer is a drug used for diabetes type 2. It was first thought that the effect of TZD was through the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR- γ). Recently the mitochondrial pyruvate carrier complex (MPC) that can be found in the mitochondrial inner membrane has been found as a target for TZD. There are two subunits of MPCs in humans; MPC1 and MPC2. The two MPCs form an active complex that transports the pyruvate into the mitochondrial matrix from the cytosol. In this work, we aimed to form the human MPC complex in vitro by overproducing His-tagged MPC1 and MPC2 in the yeast Pichia pastoris. The proteins were solubilized with n-Dodecyl-beta-Maltoside (DDM) detergent before purified with immobilized affinity chromatography (IMAC). The IMAC fractions were analyzed on SDS-PAGE and western blot with anti-His. To form the human MPC complex MPC1 was incubated with Tobacco Etch Virus (TEV) protease to cleave off the His-tag and MPC1 was then purified with reverse IMAC. The untagged MPC1 was combined with the tagged MPC2 to form the complex and purify it with IMAC followed by SDS-PAGE and western blot with anti-His and anti-MPC1. The result from the western blot shows that in the complex both MPCs are present. This paves the way for future structural and functional studies of the human MPC complex.}},
  author       = {{Lozansson, Yasmin}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Cloning, purification and optimization of the human mitochondrial pyruvate carriers}},
  year         = {{2020}},
}