Nonetheless, the presence of several interaction domains in one protein, together with possible formation of higher order structures of both scaffold proteins and transmembrane interaction partners ( Long et al., 2003 Ivarsson, 2012), strongly suggest that measurements in vitro cannot replicate the behavior of the native environment. Such approaches reduce the complexity and makes binding assays simpler to both perform and analyze. Our current understanding of the dynamics and kinetics underlying scaffold interactions relies mostly on in vitro assays of single domains isolated form their native membrane environment ( Vincentelli et al., 2015 Stiffler et al., 2007 Long et al., 2003 Ivarsson, 2012). A broad variety of different protein-protein and protein-lipid interacting domains are found in scaffold proteins, enabling them to bind and direct localization and function of their diverse interaction partners, such as receptors, transporters, ion channels and kinases ( Hung and Sheng, 2002 Zhu et al., 2016). Scaffold proteins play a key role in these efforts by operating as versatile nanoscale modules capable of bringing distinct molecular components in close proximity to shape specificity in cellular signaling networks and regulate output ( Good et al., 2011 Zeke et al., 2009). It is of fundamental importance for cell function to organize signaling processes in space and time. Together these findings imply that the ability of scaffolding proteins to bind to their partner proteins is finely tuned to encode specific responses in cells in different situations – a hypothesis that Erlendsson, Thorsen et al. Further experiments suggest that altering the ability of the PDZ domain of PICK1 to bind to partner proteins changes the mode of action of the PICK1 protein so that it can activate different responses in the cell. Unexpectedly, the experiments show that the shape and physical characteristics of the partner protein have no effect on the increase in the strength of the binding. This is due to the scaffolding proteins binding more strongly to both their partners and the membrane. The experiments show that PICK1 and PSD-95 bind to their partner proteins up to 100 times more strongly than previously observed using other approaches. used a new experimental setup known as supported cell membrane sheets – which provides direct access to the side of the cell membrane that usually faces into the cell – to study two scaffolding proteins known as PICK1 and PSD-95. To overcome this challenge, Erlendsson, Thorsen et al. However, the precise way in which scaffolding proteins assemble such groups is not clear because it is technically challenging to study this process in living cells. Previous studies have shown that scaffolding proteins are able to bind to several other proteins as well as the membrane itself at the same time. Many scaffolding proteins assemble groups of proteins on the surface of the membrane that surrounds the cell. Defects in scaffolding proteins can lead to cancer, psychiatric disorders and other diseases, so these proteins represent potential new targets for medicinal drugs. Scaffold proteins play an essential role in organizing these signals by bringing specific proteins and other molecules into close contact at particular times and locations within the cell. It is, therefore, very important that the signals are sent to the right places at the right times. Inside a cell, many different signals carry information that is essential for the cell to remain healthy and perform its role in the body. Our data supported by simulations suggest that intrinsic PDZ domain affinities are finely tuned and encode specific cellular responses, enabling multiplexed cellular functions of PDZ scaffolds. Interestingly, discrete changes in the intrinsic PICK1 PDZ affinity did not affect overall binding strength but instead revealed dual scaffold modes for PICK1. The kinetics of the binding were remarkably slow and binding strength two-three orders of magnitude higher than the intrinsic affinity for the isolated PDZ interaction. Our data demonstrate how multivalent protein-protein and protein-lipid interactions provide critical avidity for the strong binding between the PDZ domain scaffold proteins, PICK1 and PSD-95, and their cognate transmembrane binding partners. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time.
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