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Visualizing cell maintenance: Chemical tools to investigate the microenvironments of misfolded proteins

Final Report Summary - VISQC (Visualizing cell maintenance: Chemical tools to investigate the microenvironments of misfolded proteins)

Visualizing cell maintenance: Chemical tools to investigate the microenvironments of misfolded proteins

Cellular homeostasis is a process were cells maintain their internal environment optimal for proper cell functioning. Sophisticated molecular machineries carefully monitor the quality of biomolecules and remove or sequester defective ones at specific cell locations. Upon aging and disease, the capacity of the cell to maintain protein quality and function diminishes. Genetic mutations may result in protein malfunction or structural defects due to an alternation of an essential amino acid. The cell has evolved many mechanisms to handle misfolded proteins; some proteins are refolded by molecular chaperones while others are targeted for degradation by the ubiquitin proteasome system and yet others are sequestered in deposits at a specific cellular location. How cells make decisions on a specific protein destination is only hardly understood and progress in this field is slow due to the lack of useful chemical and biological tools.

Although cells are often regarded as containers that are randomly filled with biomolecules, the subcellular organization of these molecules is extremely important; biomolecules in one part might not be functional and become activated only at a distinct part of the cell by, for example, a posttranslational modification. Cells cope with environmental alterations (e.g. bacterial infection, salt concentrations, temperature) by adjusting the cellular machineries and cell-to-cell interactions. Since correct interactions between proteins are essential for many cellular processes and improper protein interactions might result in impaired cell functioning and disease, it is essential to gain information about the associating partners of a protein to understand the function within a cell and the mechanisms underlying health and disease.

To identify the permanent or dynamic protein interactions, several in vitro and in vivo techniques are available, including cell fractionation, immunoprecipitation and chemical- or photo-crosslinking. The proteins are labeled, extracted from the biological sample and then identified by mass spectrometry (Figure 1). While useful for many application it remains a challenge to identify transient, low affinity or distal interacting proteins of a specific protein of interest (POI) using these methods. In addition, these approaches often require isolation of the POI from its native cellular environment leading to the identification of many non-specific interactors.

A major contribution to the field appeared some years ago, where proximity dependent labeling (PDL) approaches were introduced. PDL is based on enzymatic tagging of potentially interacting proteins in close vicinity of the enzyme. The enzymes for the PDL strategy activate small molecules resulting in a reactive intermediate, which covalently labels the enzyme itself and proximal proteins. Local expression of the enzyme in the cells is achieved by a genetic tag encoding a specific organelle or a POI. Subsequently, proteins labeled by the spatially-restricted enzyme can be enriched and identified by MS.
The first PDL strategies that have gained valuable information about protein compositions in living cells is based on the biotin ligase (BioID) and an engineered ascorbate peroxidase (APEX). BioID is based on an engineered biotin ligase (BirA*), which converts biotin and ATP into biotin-AMP which is reactive towards nucleophiles that reside in close proximity of BirA*. While BioID is used successfully for many applications, it has limited use for temporal proteomic profiling due to the long-required labeling times (6-24 hours). In contrast, APEX generates short-lived free phenoxyl radicals by oxidizing biotin-phenol in the presence of 1 mM of hydrogen peroxide for 1 minute, that will react within < 1 ms with electron-rich amino acids. Though, the exposure of cells to high levels of peroxide (1 mM), needed for the enzymatic reaction by APEX, might cause adverse effects, such as DNA damage and the induction of a cellular stress response.

In order to avoid the use of hydrogen peroxide and still preserve fast labeling times for subcellular protein labeling, we set out to establish a novel enzymatic proximity labeling strategy using arylamine N-acetyltransferase (NAT) (Figure 1). The NAT enzyme activates chemically-synthesized aryl hydroxamic acid (AHA) probes to nitrenium ions, which react fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. We envisioned that using NAT-based PDL approach will be powerfull addition to the chemical biology toolbox that can be used to monitoring cellular processes in their native cellular environment and without the need of perturbing cofactors.

This MC-CIG project consists of two parts. First, we aimed to develop and optimize the proximity labeling approach based on the NAT system. To accomplish this, we have synthesized and evaluated several AHA probes (including the control probes) and evaluated if these can be used to label proteins on a subcellular level. We have investigated the structural and electronic properties of the probes and shown that spatial localization of the NAT enzyme to specific cellular compartments allows the labeling of proteins only in close proximity of the expressed NAT enzyme. Purification of the labelled proteins was done by the use of bioorthogonal chemistry with the reactive groups present on the synthesized hydroxamic acid probes. This enzymatic labeling strategy is generic and can be implemented to address many biological questions concerning subcellular molecular dynamics.
Second, we aimed to use the NAT approach to visualize proteins that are part of the quality control system. As a model system for misfolded proteins, we used an aggregation-prone destabilizing domain (AgDD) as these form defined aggregate structures upon removal of a binding ligand. We genetically fused several AgDD sequences to the NAT enzyme and expressed them in HEK293T cells. The AgDD-NAT constructs however, were not able to form any aggregate structures. To evaluate if this was a result of the NAT enzyme fusion, we additionally generated AgDD-APEX constructs for comparison. Upon transfection and removal from the cells, these constructs did form aggregate structures upon removal of the ligand.
As we expect that the APEX system will induce undesired cellular responses, we are currently in the process of optimizing the expression levels of the AgDD-NAT system to ensure aggregate formation in the cell. Furthermore, we are exploring the effect of alternative protein aggregates and we are currently preparing stable cell lines that express the NAT enzyme that is fused to a variety of mutants of alphaB-crystallin conferring different stabilities and aggregating behavior. Insight in the maintenance and interacting proteins of these alphaB-crystallin aggregates by the QC system will provide valuable information on the cellular dynamics and recognition of misfolded proteins and efforts on finalizing these questions are ongoing. Understanding these processes will help in the development of novel diagnostics and therapeutics for diseases that are related to misfolded proteins or to a defect in the cellular protein quality control machinery.