Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis.

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain.

Maladaptive plasticity involving increased expression of AMPA-type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell-permeable, high-affinity (~2 nM) peptide inhibitor, Tat-P4 -(C5)2 , of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat-P4 -(C5)2 disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA-receptor surface expression in vivo. Moreover, Tat-P4 -(C5)2 administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat-P4 -(C5)2 as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non-tandem protein-protein interaction domains.

PDZ Domains as Drug Targets.

Protein-protein interactions within protein networks shape the human interactome, which often is promoted by specialized protein interaction modules, such as the postsynaptic density-95 (PSD-95), discs-large, zona occludens 1 (ZO-1) (PDZ) domains. PDZ domains play a role in several cellular functions, from cell-cell communication and polarization, to regulation of protein transport and protein metabolism. PDZ domain proteins are also crucial in the formation and stability of protein complexes, establishing an important bridge between extracellular stimuli detected by transmembrane receptors and intracellular responses. PDZ domains have been suggested as promising drug targets in several diseases, ranging from neurological and oncological disorders to viral infections. In this review, the authors describe structural and genetic aspects of PDZ-containing proteins and discuss the current status of the development of small-molecule and peptide modulators of PDZ domains. An overview of potential new therapeutic interventions in PDZ-mediated protein networks is also provided.

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis.