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Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca 2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca 2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2 D509V , a Ca 2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca 2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy. Abbreviations : ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A 1 ; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Oral squamous cell carcinoma, the most common type of oral cancer, affects more than 275,000 people per year worldwide. Oral squamous cell carcinoma is very aggressive, as most patients die after 3 to 5 years post-diagnosis. The initiation and progression of oral squamous cell carcinoma are multifactorial: smoking, alcohol consumption, and human papilloma virus infection are among the causes that promote its development. Although oral squamous cell carcinoma involves abnormal growth and migration of oral epithelial cells, other cell types such as fibroblasts and immune cells form the carcinoma niche. An underlying inflammatory state within the oral tissue promotes differential stress-related responses that favor oral squamous cell carcinoma. Autophagy is an intracellular degradation process that allows cancer cells to survive under stress conditions. Autophagy degrades cellular components by sequestering them in vesicles called autophagosomes, which ultimately fuse with lysosomes. Although several autophagy markers have been associated with oral squamous cell carcinoma, it remains unclear whether up- or down-regulation of autophagy favors its progression. Autophagy levels during oral squamous cell carcinoma are both timing- and cell-specific. Here we discuss how autophagy is required to establish a new cellular microenvironment in oral squamous cell carcinoma and how autophagy drives the phenotypic change of oral squamous cell carcinoma cells by promoting crosstalk between carcinoma cells, fibroblasts, and immune cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Peña-Oyarzún, Reyes, Hernández-Cáceres, Kretschmar, Morselli, Ramirez-Sarmiento, Lavandero, Torres and Criollo.)

Synthesis of β-ionone in recombinant Saccharomyces cerevisiae is limited by the efficiency of Carotenoid Cleavage Dioxygenases (CCD), membrane-tethered enzymes catalyzing the last step in the pathway. We performed in silico design and membrane affinity analysis, focused on single-point mutations of PhCCD1 to improve membrane anchoring. The resulting constructs were tested in a β-carotene hyper-producing strain by comparing colony pigmentation against colonies transformed with native PhCCD1 and further analyzed by β-ionone quantification via RP-HPLC. Two single-point mutants increased β-ionone yields almost 3-fold when compared to native PhCCD1. We also aimed to improve substrate accessibility of PhCCD1 through the amino-terminal addition of membrane destination peptides directed towards the endoplasmic reticulum or plasma membrane. Yeast strains expressing peptide-PhCCD1 constructs showed β-ionone yields up to 4-fold higher than the strain carrying the native enzyme. Our results demonstrate that protein engineering of CCDs significantly increases the yield of β-ionone synthesized by metabolically engineered yeast., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Polycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes., Methods: Wild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations., Results: PC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca 2+ transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca 2+ stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca 2+ ATPase (SERCA) activity. An increase in outward K + currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1 R4228X manifested no suppressive effects on Kv4.3 channel activity., Conclusions: Our findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility.

Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the family of ATP-dependent vitamin kinases, several bifunctional enzymes that phosphorylate hydroxymethyl pyrimidine (HMP) and pyridoxal (PL) have been described besides enzymes specific towards HMP. To determine how bifunctionality emerged, we reconstructed the sequence of three ancestors of HMP kinases, experimentally resurrected, and assayed the enzymatic activity of their last common ancestor. The latter has ∼ 8-fold higher specificity for HMP due to a glutamine residue (Gln44) that is a key determinant of the specificity towards HMP, although it is capable of phosphorylating both substrates. These results show how a specific enzyme with catalytic promiscuity gave rise to current bifunctional enzymes., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)