Exploring Protein Turnover In Vivo

In a previous blog post, we explored how a comprehensive understanding of protein turnover dynamics can provide crucial guidance for drug discovery and development. Our first post focused primarily on the study of protein turnover in cellulo. In this approach, cells grown in culture are provided with labeled amino acids or other molecules, which are incorporated into proteins and can be tracked over time to determine protein synthesis and degradation rates. However, protein turnover can also be assessed in vivo, or in the context of living organisms. While these experiments pose unique challenges, they also offer unmatched insights into protein turnover dynamics in real biological systems. In this post, we’ll summarize key methodologies used to investigate protein turnover dynamics in vivo, highlight the benefits of in vivo turnover data in drug discovery and development, and explore how scientists can integrate these insights into their own research workflows.

How can scientists study protein turnover in vivo?

The principal method used to evaluate protein turnover – both in cellulo and in vivo – is metabolic labeling. In this technique, labeled components (most commonly, amino acids) are provided as tracers. Following their incorporation during protein synthesis, the abundance of the compounds can be tracked over time. In pulsed labeling experiments, a labeled tracer is provided for a specified time interval; in pulse-chase experiments, this is followed by the introduction of unlabeled compound in excess. Consequently, protein synthesis and degradation rates can be measured, and turnover kinetics can be determined.

In addition to radioactive isotope labeling, stable isotope labeling strategies have proven to be effective in metabolic labeling experiments. In this approach, ‘light’ and ‘heavy’ variants of a particular amino acid (or other compound) are distinguished by different molecular weights. These mass-distinguished compounds are particularly amenable to detection by mass spectrometry, which enables levels of each variant to be quantitatively determined from complex biological samples. In cellulo, these labeled compounds are provided as part of the cell culture media. Analogously, heavy stable isotopes can be provided to laboratory animals via food containing these labeled compounds. This technique is known as SILAM, or Stable Isotope Labeling in Mammals.

What are the advantages of studying protein turnover in vivo?

While cell culture experiments provide a controlled and reproducible environment for the study of protein turnover, they cannot fully recapitulate the complex, interrelated nature of living organisms. For this reason, in vivo experiments are necessary to appreciate the full range of turnover dynamics within a physiological context. In vivo assays are also the only way to discover and explore higher-level phenomena related to protein turnover, including sex- and age-specific turnover dynamics and differences in turnover associated with spatial location within a tissue or organ. Researchers have leveraged in vivo studies to assess the effects of hormones, nutritional state, and even the day-night cycle on protein turnover rates, insights which would not be possible to obtain from cell culture experiments alone.

How has in vivo protein turnover data been used to guide drug discovery?

Knowledge of in vivo protein turnover rates can support many stages of the drug discovery and development process. From evaluating a potential target and selecting an appropriate therapeutic modality to interrogating the effects of drug treatment or other perturbations, an understanding of protein synthesis and degradation rates provides critical guidance for research programs. More broadly, in vivo protein turnover studies can provide valuable insight into the mechanisms underlying disease onset and progression across a variety of therapeutic areas.

In one recent study, scientists used a SILAM approach to assess protein turnover rates in mice subjected to hypoxic, normoxic, and hyperoxic conditions. Their findings suggested that hyperoxia leads to protein destabilization that can exacerbate a range of monogenic disorders. They identified MYBBP1A as a key regulator of this response and highlighted several other potential therapeutic targets relevant to chronic hypoxia and hyperoxia. Another research group used metabolic labeling to study changes in brain protein turnover in mice treated with the antidepressant drug paroxetine. Their experiments revealed that paroxetine treatment alters hippocampal protein turnover in a region-specific manner, helping to shed light on this drug’s mechanism of action.

How can researchers access high-quality in vivo protein turnover data?

One option for researchers seeking to acquire high-quality in vivo protein turnover data is to collect it themselves through SILAM or other metabolic labeling-based techniques. However, these approaches impose significant requirements, most notably around the housing and care of laboratory animals (most commonly, mice) for a prolonged experimental period. This burden is not feasible for many groups due to its high cost and the necessity for dedicated facilities and employees. These expenses can be particularly discouraging for preliminary or exploratory studies, which are less certain to result in findings that directly translate into clinical developments.

For these reasons, it is especially valuable for scientists to access existing repositories of in vivo protein turnover data that can be plumbed for relevant insights. These resources enable tissue-specific baseline turnover rates to be obtained without the need for experimental setup, data collection, or bioinformatic analysis. One such example is the Mouse Turnover Atlas™, which provides access to high-quality proprietary turnover data from 22 murine tissues and biofluids, expertly generated and analyzed to ensure data robustness and proteome-wide coverage. Flexible access options enable researchers to acquire all data from a particular tissue type, target-specific data across multiple tissue types, or full database access. By harnessing tools like the Mouse Turnover Atlas™, scientists can obtain valuable in vivo protein turnover data on-demand to accelerate their research programs.

If you’re interested in leveraging Mouse Turnover Atlas™ – or its in cellulo equivalent, Protein Turnover Atlas™ – for your drug discovery and development research, we would love to talk to you! Send us a message and we’ll be in touch to schedule an introductory call.

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