Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli.

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His10 fusion in Escherichia coli. The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.

Ultrasound guidance for sural nerve conduction studies.

INTRODUCTION: Ultrasound can potentially identify nerves and guide recording and stimulating electrode placement for nerve conduction studies (NCS). This prospective study was performed to determine whether ultrasound guidance of sural NCS results in higher action potential amplitude, fewer stimuli required, lower stimulus strength required, and less pain experienced. METHODS: Fourteen healthy individuals underwent bilateral sural NCS, both with and without ultrasound guidance. Studies were separated by at least 48 h, and the order of testing was randomly assigned. RESULTS: Ultrasound guidance resulted in significantly fewer stimuli and lower stimuli strength required to obtain supramaximal responses (P < 0.01-0.03). Ultrasound guidance required significantly more time to perform than standard sural NCS (P < 0.01). There was no difference in sural nerve amplitude or pain rating between the 2 groups. DISCUSSION: Neuromuscular ultrasound can be used effectively to guide electrode placement during sural NCS. Muscle Nerve 59:705-707, 2019.

Cardiomyocyte-specific Loss of Glutamyl-prolyl-tRNA Synthetase Leads to Disturbed Protein Homeostasis and Dilated Cardiomyopathy.

Glutamyl-prolyl-tRNA synthetase (EPRS1), an aminoacyl-tRNA synthetase (ARS) ligating glutamic acid and proline to their corresponding tRNAs, plays an essential role in decoding proline codons during translation elongation. The physiological function of EPRS1 in cardiomyocytes (CMs) and the potential effects of CM-specific loss of EPRS1 remain unknown. Here, we found that heterozygous Eprs1 knockout in CMs does not cause any significant changes in CM hypertrophy induced by pressure overload, while homozygous knockout leads to dilated cardiomyopathy, heart failure, and lethality at around 1 month after Eprs1 deletion. Transcriptomic profiling of early-stage Eprs1 knockout hearts suggests a significantly decreased expression of multiple ion channel genes and an increased gene expression in proapoptotic pathways and integrated stress response. Proteomic analysis shows decreased protein expression of multi-aminoacyl-tRNA synthetase complex components, fatty acid, and branched-chain amino acid metabolic enzymes, as well as a compensatory increase in cytosolic translation machine-related proteins. Immunoblot analysis indicated that multiple proline-rich proteins were reduced at the early stage, which might contribute to cardiac dysfunction of Eprs1 knockout mice. Taken together, this study demonstrates the physiological and molecular outcome of loss-of-function of EPRS1 in vivo and provides valuable insights into the potential side effects on CMs resulting from the EPRS1-targeting therapeutic approach.

Cardiomyocyte-Specific Loss of Glutamyl-prolyl-tRNA Synthetase Leads to Disturbed Protein Homeostasis and Dilated Cardiomyopathy.

Glutamyl-prolyl-tRNA synthetase (EPRS1), an aminoacyl-tRNA synthetase (ARS) ligating glutamic acid and proline to their corresponding tRNAs, plays an essential role in decoding proline codons during translation elongation. The physiological function of EPRS1 in cardiomyocytes (CMs) and the potential effects of the CM-specific loss of Eprs1 remain unknown. Here, we found that heterozygous Eprs1 knockout in CMs does not cause any significant changes in CM hypertrophy induced by pressure overload, while homozygous knockout leads to dilated cardiomyopathy, heart failure, and lethality at around 1 month after Eprs1 deletion. The transcriptomic profiling of early-stage Eprs1 knockout hearts suggests a significantly decreased expression of multiple ion channel genes and an increased gene expression in proapoptotic pathways and integrated stress response. Proteomic analysis shows decreased protein expression in multi-aminoacyl-tRNA synthetase complex components, fatty acids, and branched-chain amino acid metabolic enzymes, as well as a compensatory increase in cytosolic translation machine-related proteins. Immunoblot analysis indicates that multiple proline-rich proteins were reduced at the early stage, which might contribute to the cardiac dysfunction of Eprs1 knockout mice. Taken together, this study demonstrates the physiological and molecular outcomes of loss-of-function of Eprs1 in vivo and provides valuable insights into the potential side effects on CMs, resulting from the EPRS1-targeting therapeutic approach.

Expression and purification of the mitochondrial transmembrane protein FAM210A in Escherichia coli.

The protein Family with sequence similarity 210 member A (FAM210A) is a mitochondrial inner membrane protein that regulates the protein synthesis of mitochondrial DNA encoded genes. However, how it functions in this process is not well understood. Developing and optimizing a protein purification strategy will facilitate biochemical and structural studies of FAM210A. Here, we developed a method to purify human FAM210A with deleted mitochondrial targeting signal sequence using the MBP-His 10 fusion in Escherichia coli . The recombinant FAM210A protein was inserted into the E. coli cell membrane and purified from isolated bacterial cell membranes, followed by a two-step process using Ni-NTA resin-based immobilized-metal affinity chromatography (IMAC) and ion exchange purification. A pulldown assay validated the functionality of purified FAM210A protein interacting with human mitochondrial elongation factor EF-Tu in HEK293T cell lysates. Taken together, this study developed a method for purification of the mitochondrial transmembrane protein FAM210A partially complexed with E.coli derived EF-Tu and provides an opportunity for future potential biochemical and structural studies of recombinant FAM210A protein.