Disease-Related Protein Variants of the Highly Conserved Enzyme PAPSS2 Show Marginal Stability and Aggregation in Cells.

Cellular sulfation pathways rely on the activated sulfate 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In humans, PAPS is exclusively provided by the two PAPS synthases PAPSS1 and PAPSS2. Mutations found in the PAPSS2 gene result in severe disease states such as bone dysplasia, androgen excess and polycystic ovary syndrome. The APS kinase domain of PAPSS2 catalyzes the rate-limiting step in PAPS biosynthesis. In this study, we show that clinically described disease mutations located in the naturally fragile APS kinase domain are associated either with its destabilization and aggregation or its deactivation. Our findings provide novel insights into possible molecular mechanisms that could give rise to disease phenotypes associated with sulfation pathway genes.

Cellular ATP Levels Determine the Stability of a Nucleotide Kinase.

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf 0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

Quantifying the thermodynamics of protein unfolding using 2D NMR spectroscopy.

A topic that has attracted considerable interest in recent years is the possibility to perform thermodynamic studies of proteins directly in-cell or in complex environments which mimic the cellular interior. Nuclear magnetic resonance (NMR) could be an attractive technique for these studies but its applicability has so far been limited by technical issues. Here, we demonstrate that 2D NMR methods can be successfully applied to measure thermodynamic parameters provided that a suitable choice of the residues used for the calculation is made. We propose a new parameter, named RAD, which reflects the level of protection of a specific amide proton in the protein core and can guide through the selection of the resonances. We also suggest a way to calibrate the volumes to become independent of technical limitations. The methodology we propose leads to stability curves comparable to that calculated from CD data and provides a new tool for thermodynamic measurements in complex environments.

Magnetic spatiotemporal control of SOS1 coupled nanoparticles for guided neurite growth in dopaminergic single cells.

The axon regeneration of neurons in the brain can be enhanced by activating intracellular signaling pathways such as those triggered by the membrane-anchored Rat sarcoma (RAS) proto-oncogene. Here we demonstrate the induction of neurite growth by expressing tagged permanently active Harvey-RAS protein or the RAS-activating catalytic domain of the guanine nucleotide exchange factor (SOS1cat), in secondary dopaminergic cells. Due to the tag, the expressed fusion protein is captured by functionalized magnetic nanoparticles in the cytoplasm of the cell. We use magnetic tips for remote translocation of the SOS1cat-loaded magnetic nanoparticles from the cytoplasm towards the inner face of the plasma membrane where the endogenous Harvey-RAS protein is located. Furthermore, we show the magnetic transport of SOS1cat-bound nanoparticles from the cytoplasm into the neurite until they accumulate at its tip on a time scale of minutes. In order to scale-up from single cells, we show the cytoplasmic delivery of the magnetic nanoparticles into large numbers of cells without changing the cellular response to nerve growth factor. These results will serve as an initial step to develop tools for refining cell replacement therapies based on grafted human induced dopaminergic neurons loaded with functionalized magnetic nanoparticles in Parkinson model systems.

Melting Down Protein Stability: PAPS Synthase 2 in Patients and in a Cellular Environment.

Within the crowded and complex environment of the cell, a protein experiences stabilizing excluded-volume effects and destabilizing quinary interactions with other proteins. Which of these prevail, needs to be determined on a case-by-case basis. PAPS synthases are dimeric and bifunctional enzymes, providing activated sulfate in the form of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for sulfation reactions. The human PAPS synthases PAPSS1 and PAPSS2 differ significantly in their protein stability as PAPSS2 is a naturally fragile protein. PAPS synthases bind a series of nucleotide ligands and some of them markedly stabilize these proteins. PAPS synthases are of biomedical relevance as destabilizing point mutations give rise to several pathologies. Genetic defects in PAPSS2 have been linked to bone and cartilage malformations as well as a steroid sulfation defect. All this makes PAPS synthases ideal to study protein unfolding, ligand binding, and the stabilizing and destabilizing factors in their cellular environment. This review provides an overview on current concepts of protein folding and stability and links this with our current understanding of the different disease mechanisms of PAPSS2-related pathologies with perspectives for future research and application.

Imperfect crowding adaptation of mammalian cells towards osmotic stress and its modulation by osmolytes.

Changes of the extracellular milieu could affect cellular crowding. To prevent detrimental effects, cells use adaptation mechanisms to react to such conditions. Using fluorescent crowding sensors, we show that the initial response to osmotic stress is fast but imperfect, while the slow response renders cells more tolerant to stress, particularly in the presence of osmolytes.

Elucidation of the Catalytic Mechanism of a Miniature Zinc Finger Hydrolase.

To improve our mechanistic understanding of zinc metalloenzymes, we report a joint computational and experimental study of a minimal carbonic anhydrase (CA) mimic, a 22-residue Zn-finger hydrolase. We combine classical molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) geometry optimizations, and QM/MM free energy simulations with ambient and high-pressure kinetic measurements to investigate the mechanism of the hydrolysis of the substrate p-nitrophenylacetate (pNPA). The zinc center of the hydrolase prefers a pentacoordinated geometry, as found in most naturally occurring CAs and CA-like enzymes. Two possible mechanisms for the catalytic reaction are investigated. The first one is analogous to the commonly accepted mechanism for CA-like enzymes: a sequential pathway, in which a Zn2+-bound hydroxide acts as a nucleophile and the hydrolysis proceeds through a tetrahedral intermediate. The initial rate-limiting step of this reaction is the nucleophilic attack of the hydroxide on pNPA to form the tetrahedral intermediate. The computed free energy barrier of 18.5 kcal/mol is consistent with the experimental value of 20.5 kcal/mol obtained from our kinetics experiments. We also explore an alternative reverse protonation pathway for the hydrolase, in which a nearby hydroxide ion from the bulk acts as the nucleophile (instead of a zinc-bound hydroxide). According to QM/MM MD simulations, hydrolysis occurs spontaneously along this pathway. However, this second scenario is not viable in our system, as the tertiary structure of the hydrolase lacks a suitably positioned residue that would act as a general base and generate a hydroxide ion from a nearby bulk water molecule. Hence, our combined theoretical and experimental study indicates that the investigated minimal CA mimic retains the essential mechanistic features of CA-like enzyme catalysis. The high-pressure experiments show that its catalytic efficiency can be enhanced by applying hydrostatic pressure. According to the simulations, more drastic improvements might be afforded by mutations that make the reverse protonation pathway accessible.

Excluded-volume effects in living cells.

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.