Protein Stability In Cells Research Articles

Unraveling the enthalpy of macromolecular crowding.

Concentrated solutions of synthetic polymers are often used to stabilize proteins, but there are few informed models that provide a detailed explanation of the effect. Classic crowding models are based on spherical, colloid-based solution theories, but such models only account for steric effects. To aid in the establishment of new, more informed, model, we acquired an extensive dataset that quantifies the effect of polyethylene glycol molecular weight and concentration on the temperature dependence of protein stability using 19F NMR. More specifically, we investigated the stability of the 7-kDa metastable N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) in polyethylene glycol solutions and quantified the modified standard-state free energy of unfolding and its enthalpic and entropic components. We used these data to build a new model which shows that interactions of solution components with both the folded and unfolded states must be considered. With this more nuanced understanding, we can better predict how polymers will affect the stability of protein-based drugs, how different biological molecules impact protein stability in cells and perhaps inform the design of even more useful stabilizing agents.

An overview of heat-stress response regulation in Gram-negative bacteria considering Escherichia coli as a model organism

Response to heat stress (HSR) is a key stress response for endurance in Escherichia coli mediated by transcriptional factor σ-32. Even though there has been extensive investigation on the contribution of proteins and chaperones in retaining protein stability in cells under stress conditions, limited information is available regarding the dynamic nature of mechanisms regulating the activity of the highly conserved heat shock proteins (Hsps). Several gene expression-based studies suggest the pivotal role of Hsp70 (DnaK) in the regulation of the expression of heat shock genes (Hsg). Direct interaction of Hsp70 with σ-32 may regulate this function in E. coli. Recent studies revealed that localization of σ-32 to the membrane interior by SRP-dependent pathway enables them to function appropriately in their role as regulators. The contributions of different cellular components including cell membrane remain unknown. Other cellular components or σ-32 interfere with polypeptides which could play a crucial role in cell survival. Sigma factor monitors and preserves outer membrane integrity of E. coli by stimulating the genes regulating outer membrane proteins (OMPs) and lipopolysaccharide (LPS) assemblage as well as through expression of small RNAs to down-regulate surplus unassembled OMPs. σ-E activity is regulated by the rate at which its membrane-encompassing anti-sigma factor, RseA is degraded. Mutations in rseA are reported to constitutively increase the sigma (E) activity that is validated at both genetic and biochemical levels. In this review, the basic mechanism of heat stress regulation in gram-negative bacteria has been elaborated using E. coli as a model organism.

USP12 promotes breast cancer angiogenesis by maintaining midkine stability

Deubiquitinases (DUBs) have important biological functions, but their roles in breast cancer metastasis are not completely clear. In this study, through screening a series of DUBs related to breast cancer distant metastasis-free survival (DMFS) in the Kaplan-Meier Plotter database, we identified ubiquitin-specific protease 12 (USP12) as a key deubiquitinating enzyme for breast cancer metastasis. We confirmed this via an orthotopic mouse lung metastasis model. We revealed that the DMFS of breast cancer patients with high USP12 was worse than that of others. Knockdown of USP12 decreased the lung metastasis ability of 4T1 cells, while USP12 overexpression increased the lung metastasis ability of these cells in vivo. Furthermore, our results showed that the supernatant from USP12-overexpressing breast cancer cells could promote angiogenesis according to human umbilical vein endothelial cell (HUVEC) migration and tube formation assays. Subsequently, we identified midkine (MDK) as one of its substrates. USP12 could directly interact with MDK, decrease its polyubiquitination and increase its protein stability in cells. Overexpression of MDK rescued the loss of angiogenesis ability mediated by knockdown of USP12 in breast cancer cells in vitro and in vivo. There was a strong positive relationship between USP12 and MDK protein expression in clinical breast cancer samples. Consistent with the pattern for USP12, high MDK expression predicted lower DMFS and overall survival (OS) in breast cancer. Collectively, our study identified that USP12 is responsible for deubiquitinating and stabilizing MDK and leads to metastasis by promoting angiogenesis. Therefore, the USP12–MDK axis could serve as a potential target for the therapeutic treatment of breast cancer metastasis.

An enzyme-based biosensor for monitoring and engineering protein stability in vivo

Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.

The intracellular environment affects protein–protein interactions

Protein-protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, Escherichia coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. coli Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded than E. coli cells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous-and specific-interactions coherently evolve for a protein to properly function in the crowded cellular environment.

Recent advances in bioanalytical methods to measure proteome stability in cells.

Proteome stability constitutes an essential aspect of protein homeostasis (proteostasis). Proteostasis networks maintain proteins and their interactors in a defined conformation for their activity, localisation, and function. However, endogenous or exogenous stressors can perturb proteostasis integrity and deplete folding capacity, generating destabilized folding intermediates and deleterious aggregated species. Over the years, protein unfolding, misfolding and aggregation have been reported to be associated with aging and many diseases such as neurodegenerative diseases, diabetes, cardiac disease and toxicity, and cancers. Therefore, monitoring proteome stability is central to understanding underlying biological processes and mechanisms of disease progression. Herein, we review the recent bioanalytical methods to measure protein stability in cells on a proteome-wide scale.

Photoactive Bifunctional Degraders: Precision Tools To Regulate Protein Stability.

Targeted protein degradation with bifunctional degraders is positioned as a remarkable game-changing strategy to control cellular protein levels and promises a new therapeutic modality in drug discovery. Light activation of a degrader to achieve exquisite spatiotemporal control over protein stability in cells has attracted the interest of multiple research groups, with recent reports demonstrating optical control of proteolysis with chimeric molecules bearing photolabile or photoswitchable motifs. In this context of targeted proteolysis research spurring the emergence of innovative tools, we examine the design, synthesis, and properties of light-activated degraders. The significant impact of this approach in regulating disease-relevant protein levels in a light-dependent manner is highlighted with key examples, and future developments to fully harness the potential of light-induced protein degradation with photoactive bifunctional molecules are discussed.

SCF-FBXO24 regulates cell proliferation by mediating ubiquitination and degradation of PRMT6

The protein arginine methyltransferase 6 (PRMT6) is a coregulator of gene expression by methylation of the histone H3 on arginine 2 (H3R2), H4R3 and H2AR3 [1,2]. PRMT6 is aberrantly expressed in various types of human cancer, and abnormal methylation in cancers caused by overexpression of PRMT6 is considered to correlate with poor recovery prognosis [3,4]. However, mechanisms that regulate PRMT6 protein stability in cells remain largely unknown. Here we identified that an orphan F-box protein, FBXO24, that binds to 270 to 275 amino acid residues of PRMT6 to cause polyubiquitination of lysine at position 369 of PRMT6, which mediates its degradation via the ubiquitin-proteasome pathway. Overexpression of FBXO24 or knockout of PRMT6 was found to inhibit cell proliferation, migration, and invasion in H1299 cells. PRMT6 K369R mutant became resistant to degradation. Overexpression of PRMT6 K369R caused cell cycle progression, resulting in cell proliferation. Thus, our data confirm that FBXO24 regulates cell proliferation by mediating ubiquitin-dependent proteasomal degradation of PRMT6.

Identification of Miro1 and Miro2 as mitochondrial receptors for myosin XIX

Mitochondrial distribution in cells is critical for cellular function and proper inheritance during cell division. In mammalian cells, mitochondria are transported predominantly along microtubules by kinesin and dynein motors that bind indirectly via TRAK1 and TRAK2 to outer mitochondrial membrane proteins Miro1 and Miro2 (Miro1/2). Here, using proximity labelling, we identified Miro1/2 as potential binding partners of myosin XIX (Myo19). Interaction studies show that Miro1 binds directly to a C-terminal fragment of the Myo19 tail region and that Miro1/2 recruit the Myo19 tail in vivo This recruitment is regulated by the nucleotide state of the N-terminal Rho-like GTPase domain of Miro1/2. Notably, Myo19 protein stability in cells depends on its association with Miro1/2. Downregulation of Miro1/2 or overexpression of the adaptor proteins TRAK1 and TRAK2 caused a reduction in Myo19 protein levels. Myo19 regulates the subcellular distribution of mitochondria, and downregulation, as well as overexpression, of Myo19 induced perinuclear collapse of mitochondria, phenocopying loss of the kinesin KIF5, dynein or their mitochondrial receptors Miro1/2. These results suggest that Miro1 and Miro2 coordinate microtubule- and actin-based mitochondrial movement.This article has an associated First Person interview with the first author of the paper.

Non-Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells.

Although biomolecules evolved to function in the cell, most biochemical assays are carried out in vitro. In-cell studies highlight how steric and non-steric interactions modulate protein folding and interactions. VlsE and PGK present two extremes of chemical behavior in the cell: the extracellular protein VlsE is destabilized in eukaryotic cells, whereas the cytoplasmic protein PGK is stabilized. VlsE and PGK are benchmarks in a systematic series of solvation environments to distinguish contributions from non-steric and steric interactions to protein stability, compactness, and folding rate by comparing cell lysate, a crowding agent, ionic buffer and lysate buffer with in-cell results. As anticipated, crowding stabilizes proteins, causes compaction, and can speed folding. Protein flexibility determines its sensitivity to steric interactions or crowding. Non-steric interactions alone predict in-cell stability trends, while crowding provides an offset towards greater stabilization. We suggest that a simple combination of lysis buffer and Ficoll is an effective new in vitro mimic of the intracellular environment on protein folding and stability.

Osmotic Shock Induced Protein Destabilization in Living Cells and Its Reversal by Glycine Betaine

Many organisms can adapt to changes in the solute content of their surroundings (i.e., the osmolarity). Hyperosmotic shock causes water efflux and a concomitant reduction in cell volume, which is countered by the accumulation of osmolytes. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase protein stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living Escherichia coli cells before and after hyperosmotic shock in the presence and absence of the osmolyte, glycine betaine. The 7-kDa N-terminal src-homology 3 domain of Drosophila signal transduction protein drk is used as the test protein. We find that hyperosmotic shock decreases SH3 stability in cells, consistent with the idea that transient attractive interactions are important under physiologically relevant crowded conditions. The subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient attractive interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.

Glycine Betaine Reverses Osmotic Shock Induced Protein Destabilization in Living Cells

Cells utilize several mechanisms for adapting to changes in osmotic pressure. Bacteria, including Escherichia coli, can grow in a wide range of osmolarities. Increasing the external osmolarity (i.e., hyperosmotic shock) causes water efflux, reduction in cell volume and accumulation of osmolytes such as glycine betaine. This volume reduction increases the crowded nature of the cytoplasm, which is expected to affect protein stability. In contrast to traditional theory, which predicts that more crowded conditions can only increase stability, recent work shows that crowding can destabilize proteins through transient attractive interactions. Here, we quantify protein stability in living E. coli cells before and after osmotic shock in the presence and absence of glycine betaine. The 7-kDa N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) is used as the model protein because it exists in an equilibrium between a folded state and an unfolded ensemble. Labeling SH3 with a fluorine on its sole tryptophan facilitates NMR-based detection of both states simultaneously, allowing quantification of the free energy of unfolding in vitro and in living E. coli cells. We find that hyperosmotic shock decreases SH3 stability, consistent with the idea that weak interactions are important under physiologically relevant crowded conditions. Subsequent uptake of glycine betaine returns SH3 to the stability observed without osmotic shock. These results highlight the effect of transient interactions on protein stability in cells and provide a new explanation for why stressed cells accumulate osmolytes.

The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive.

It is frequently assumed that fluorescent protein tags used in biological imaging experiments are minimally perturbing to their host protein. As in-cell experiments become more quantitative and measure rates and equilibrium constants, rather than just "on-off" activity or the presence of a protein, it becomes more important to understand such perturbations. One criterion for a protein modification to be a perturbation is additivity of two perturbations (a linear effect on the protein free energy). Here we show that adding fluorescent protein tags to a host protein in vitro has a large nonadditive effect on its folding free energy. We compare an unlabeled, three singly labeled, and a doubly labeled enzyme (phosphoglycerate kinase). We propose two mechanisms for nonadditivity. In the "quinary interaction" mechanism, two tags interact transiently with one another, relieving the host protein from unfavorable tag-protein interactions. In the "crowding" mechanism, adding two tags provides the minimal crowding necessary to overcome destabilizing interactions of individual tags with the host protein. Both of these mechanisms affect protein stability in cells; we show here that they must also be considered for tagged proteins used for reference in vitro.

Crowding and Protein Dimerization

In cells, proteins are surround by macromolecules at concentrations of greater than 100 g/L, yet the majority of our knowledge comes from experiments conducted in dilute buffer solutions. The structure and stability of proteins in cells is influenced by two interactions, hard core repulsions, which arise from excluded volume effects, and transient chemical interactions with surrounding molecules. High concentrations of inert polymers, small molecule cosolutes, and proteins are often used to mimic cellular conditions. Here, we describe the effects of crowding on a protein-protein interaction by using 19F NMR spectroscopy and a variant of the 6 kDa globular B1 domain of protein G. The A34F variant was previously shown to form a side by side dimer in buffer. Using the 3-flourotyorsine labeled variant we measured a dissociation constant of 59 ± 5 μM, consistent with previous the study. We then proceed to show the influences of co-solutes on dimer dissociation. Crowding agents such as sucrose, Ficoll-70, glycine betaine, trimethylamineoxide, and bovine serum albumin stabilize the dimer, whereas urea, ethylene glycol, 8 kDa polyethylene glycol and lysozyme destabilize the dimer. Measuring the temperature dependence of dimerization allows for us to measure the van’t Hoff enthalpy of dimer formation. We are now extending this methodology to in-cell NMR studies of the dimer.

Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability.

G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner.

Structure of a BAG6 (Bcl-2-associated Athanogene 6)-Ubl4a (Ubiquitin-like Protein 4a) Complex Reveals a Novel Binding Interface That Functions in Tail-anchored Protein Biogenesis

BAG6 is an essential protein that functions in two distinct biological pathways, ubiquitin-mediated protein degradation of defective polypeptides and tail-anchored (TA) transmembrane protein biogenesis in mammals, although its structural and functional properties remain unknown. We solved a crystal structure of the C-terminal heterodimerization domains of BAG6 and Ubl4a and characterized their interaction biochemically. Unexpectedly, the specificity and structure of the C terminus of BAG6, which was previously classified as a BAG domain, were completely distinct from those of the canonical BAG domain. Furthermore, the tight association of BAG6 and Ubl4a resulted in modulation of Ubl4a protein stability in cells. Therefore, we propose to designate the Ubl4a-binding region of BAG6 as the novel BAG-similar (BAGS) domain. The structure of Ubl4a, which interacts with BAG6, is similar to the yeast homologue Get5, which forms a homodimer. These observations indicate that the BAGS domain of BAG6 promotes the TA protein biogenesis pathway in mammals by the interaction with Ubl4a.

Quinary structure modulates protein stability in cells

Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein-matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli. We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome.

Quinary structure modulates protein stability in cells

Quinary structure modulates protein stability in cells

Regulation of Mdm2 protein stability and the p53 response by NEDD4-1 E3 ligase

Mdm2 is a critical negative regulator of the tumor suppressor protein p53. Mdm2 is an E3 ligase whose overexpression leads to functional inactivation of p53. Mdm2 protein stability is regulated by several mechanisms including RING (Really Interesting New Gene) domain-mediated autoubiquitination. Here we report biochemical identification of NEDD4-1 as an E3 ligase for Mdm2 that contributes to the regulation of Mdm2 protein stability in cells. NEDD4-1 was identified from Jurkat cytosolic fractions using an enzyme-dead Mdm2 mutant protein as a substrate for in vitro E3 ligase assays. We show that lysates from Nedd4-1 knockout (KO) mouse embryonic fibroblasts (MEFs) have significantly diminished E3 ligase activity toward Mdm2 compared with lysates from wild-type (WT) MEFs. Recombinant NEDD4-1 promotes Mdm2 ubiquitination in vitro in a concentration- and time-dependent manner. In cells, NEDD4-1 physically interacts with Mdm2 via the RING domain of Mdm2. Overexpression of NEDD4-1, but not an enzyme-dead NEDD4-1CS mutant, increases ubiquitination of Mdm2. NEDD4-1 catalyzes the formation of K63-type polyubiquitin chains on Mdm2 that are distinct from K48-type polyubiquitination chains mediated by the Mdm2/MdmX complex. Importantly, K63-type polyubiquitination by NEDD4-1 competes with K48-type polyubiquitination on Mdm2 in cells. As a result, NEDD4-1-mediated ubiquitination stabilizes Mdm2. NEDD4-1 knockdown reduces the t1/2 (half-life) of endogenous Mdm2 from 20 to 12 min in U2OS cells. Nedd4-1 KO MEFs manifest increased p53 levels and activity, a more robust DNA damage response and increased G1 arrest compared with WT MEFs. Similarly, NEDD4-1 knockdown in WT-p53-bearing cells increases basal p53 levels and activity in an Mdm2-dependent manner, causes stronger p53 responses to DNA damage and results in p53-dependent growth inhibition compared with corresponding NEDD4-1-proficient control cells. This study identifies NEDD4-1 as a novel component of the p53/Mdm2 regulatory feedback loop that controls p53 activity during stress responses.

Protein Stability in Living Cells

Theories concerning the effects of macromolecular crowding assert that the biophysical properties of proteins and nucleic acids can be significantly altered in native cellular environments relative to buffer alone. Despite a growing number of studies probing equilibrium thermodynamic protein stability in cells, there remains a lack of quantitative information, especially regarding residue-level stability under non-perturbing conditions. We have measured the in-cell stability of the 56-amino acid B1 domain of protein G (GB1) at the residue level without using destabilizing solutes or thermal modification by using NMR-detected hydrogen-deuterium exchange of quenched cell lysates. Comparison to dilute solution (pH 7.6 and 37 °C) shows that residues are stabilized in Escherichia coli cells by as much as 1.1 ±0.1 kcal/mol (Figure 1). We have also identified the residues most important for global folding of GB1 in cells. We discuss the implications of our findings with respect to structural models gleaned from studies conducted in buffer alone.View Large Image | View Hi-Res Image | Download PowerPoint Slide