Naturally Occurring Osmolytes Modulate the Nanomechanical Properties of Polycystic Kidney Disease Domains

Abstract

Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1. Polycystin-1 (PC1)2 is a large transmembrane protein, which, when mutated, cause autosomal dominant polycystic kidney disease (PKD), one of the most common lifethreatening genetic diseases, which is a leading cause of kidney failure (1). The available evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary (luminal) cilia, neighboring cells, and extracellular matrix and transduces them into cellular responses that regulate proliferation, adhesion, and differentiation that are essential for the control of renal tubules and kidney morphogenesis (1-4).PC1 is expressed along the renal tubule, where it is exposed to a wide range of concentrations of urea, from 5 mM in the proximal tubule to up to ϳ1 M in the collecting duct (5-7). Urea is known as a common denaturant that affects protein function by perturbing their structure (8). Several osmolytes have been found in the mammalian kidney that are known to counteract the effects of urea on proteins (9 -15). Among epithelial cells of the kidney medulla, the principal organic osmolytes include the polyols sorbitol and inositol, the methylamines betaine and glycerophosphocholine, and the amino acid taurine. Protecting osmolytes are found in all taxa (11,14). For example, cartilaginous fish concentrate trimethylamine N-oxide (TMAO), to offset the damaging effects of urea on protein function, and it is one of the best studied protecting osmolytes (e.g. 16 -20).The mecha...

Probing osmolyte participation in the unfolding transition state of a protein

“…Denaturing and protecting osmolytes therefore offer an attractive route to modulate the mechanical properties of a protein. Indeed, recent single-molecule atomic force microscopy (AFM) experiments have shown that naturally occurring protecting and denaturing osmolytes have profound effects on the mechanical folding pathways of polycystic kidney disease (PKD) domains (37). This study demonstrated that protecting osmolytes such as sorbitol and trimethylamine N oxide are efficient in counteracting the effect of the denaturing osmolyte urea on the mechanical stability of PKD domains.…”

Protein folding and stability in the presence of osmolytes

“…In turn, mammalian liver cells, besides urea, contain myo-inositol, sorbitol, betaine, taurine, glycerylphosphorylcholine, and a number of polyols and methylamines [4,[117][118][119]. The functions of neutralizing osmolytes in cells that contain a mix of urea and osmoprotectors, which consist of opposing the denaturing effect of urea and the recovery of the structure and functional activity of the protein, are sufficiently clear [20,120,121]. This leads to the question of how independent the protein effects of individual osmolytes are.…”

Polycystins, focal adhesions and extracellular matrix interactions

“…The step-wise unfolding of Polycystin-1 has been linked to the tandemly repeated PKD repeats that unfold sequentially under stretch [68]. The distensibility of Polycystin 1 is modulated by disease-causing mutations and environmental conditions [70, 71] suggesting that force-induced changes in Polycystin-1 structure may be physiologically relevant. While the current data support the idea that Polycystin-1 can function as a flexible and elastic linkage between cells or between cells and the extracellular matrix, future studies will have to accommodate the findings that the entire Polycystin-1 extracellular domain is autocatalytically cleaved from its transmembrane spanning domains and that cleavage is required for at least some of Polycystin-1 post-natal functions [72].…”