Quantification Of Protein Synthesis Research Articles

Visualisation of in vivo protein synthesis during mycobacterial infection through [68Ga]Ga-DOTA-puromycin µPET/MRI

Radiolabelled puromycin analogues will allow the quantification of protein synthesis through nuclear medicine-based imaging. A particularly useful application could be the non-invasive longitudinal visualisation of mycobacterial activity through direct quantification of puromycin binding. This study assesses the value of [68Ga]Ga-DOTA-puromycin in the visualisation of mycobacteria through positron emission tomography combined with magnetic resonance imaging (µPET/MRI). The radiopharmaceutical was produced by previously published and validated methods. [68Ga]Ga-DOTA-Puromycin imaging was performed on severe immunodeficient mice infected with Bacille Calmette-Guérin-derived M. Bovis (BCG). Acute and chronic infection stages were examined by µPET/MRI. A follow-up group of animals acted as controls (animals bearing S. aureus-derived infection and sterile inflammation) to assess tracer selectivity. [68Ga]Ga-DOTA-puromycin-µPET/MRI images revealed the acute, widespread infection within the right upper shoulder and armpit. Also, [68Ga]Ga-DOTA-puromycin signal sensitivity measured after a 12-week period was lower than that of [18F]FDG-PET in the same animals. A suitable correlation between normalised uptake values (NUV) and gold standard histopathological analysis confirms accurate tracer accumulation in viable bacteria. The radiopharmaceutical showed infection selectivity over inflammation but accumulated in both M. Bovis and S. Aureus, lacking pathogen specificity. Overall, [68Ga]Ga-DOTA-puromycin exhibits potential as a tool for non-invasive protein synthesis visualization, albeit without pathogen selectivity.

Dynamics of Non-Canonical Amino Acid-Labeled Intra- and Extracellular Proteins in the Developing Mouse.

Mapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative in vivo characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development. Pregnant C57BL/6 mice were injected with Aha or PBS (control) at different embryonic time points. Aha-labeled proteins from homogenized E12.5 and E15.5 embryos were conjugated with diazo biotin-alkyne, bound to NeutrAvidin beads, selectively released, then processed for either SDS-PAGE or LC-MS/MS. For turnover studies, embryos were harvested 0-48h after Aha injection at E12.5, separated into different cellular fractions based on solubility, and analyzed via western blotting. We developed an enhanced method for isolating Aha-labeled proteins from embryos that minimizes background signal from unlabeled proteins and avidin contamination. Approximately 50% of all identified proteins were found only in Aha samples. Comparing proteins present in both Aha and PBS samples, 90% were > 2-fold enriched in Aha-treated embryos. Furthermore, this method could resolve differences in the Aha-labeled proteome between developmental time points. Newly synthesized Aha-labeled proteins were observed by 3h and peak labeling was around 6h. Notably, extracellular matrix and cytoskeletal turnover appeared lower than the cytosolic fraction. The methods developed in this work enable the identification and quantification of protein synthesis and turnover in different tissue fractions during development. This will provide insight into functional tissue assembly and ultimately inform the design of regenerative therapies that seek to promote growth and repair.

Genome-wide identification and quantification of protein synthesis in cultured cells and whole tissues by puromycin-associated nascent chain proteomics (PUNCH-P)

Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or whole tissues by using a biotinylated derivative of puromycin, which becomes incorporated into nascent polypeptide chains by ribosome catalysis. In this method, termed puromycin-associated nascent chain proteomics (PUNCH-P), intact ribosome-nascent chain complexes are first recovered from cells by ultracentrifugation, followed by biotin-puromycin labeling of newly synthesized proteins, streptavidin affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unlike methods that require in vivo labeling, the sensitivity and coverage of PUNCH-P depend only on the amount of starting material and not on the duration of labeling, thus enabling the measurement of rapid fluctuations in protein synthesis. The protocol requires 3 d for sample preparation and analysis.

Strategies towards a better understanding of antibiotic action: folate pathway inhibition in Haemophilus influenzae as an example.

Two-dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.