Collagen IV deficiency causes hypertrophic remodeling and endothelium-dependent hyperpolarization in small vessel disease with intracerebral hemorrhage.

BACKGROUND: Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. METHODS: Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. FINDINGS: Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. INTERPRETATION: COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. FUNDING: MRC, Wellcome Trust, BHF.

Acquired Risk Factors that Influence Risk for Dementia and Possibly Alzheimer Disease.

Various medicinal agents aimed at improving Alzheimer disease (AD) include cholinesterase inhibitors, memantine, aducanumab, and antioxidants. These medications are typically prescribed once AD is diagnosed in the clinical setting in order to slow progression. Though initiating treatment after being diagnosed with AD is important, significance should be placed on recognizing known acquired risk factors in order to potentially decrease the likelihood of developing dementia and perhaps specifically AD. This article summarizes the acquired factors that influence risk for dementia.

Elevated Na is a dynamic and reversible modulator of mitochondrial metabolism in the heart.

Elevated intracellular sodium Nai adversely affects mitochondrial metabolism and is a common feature of heart failure. The reversibility of acute Na induced metabolic changes is evaluated in Langendorff perfused rat hearts using the Na/K ATPase inhibitor ouabain and the myosin-uncoupler para-aminoblebbistatin to maintain constant energetic demand. Elevated Nai decreases Gibb's free energy of ATP hydrolysis, increases the TCA cycle intermediates succinate and fumarate, decreases ETC activity at Complexes I, II and III, and causes a redox shift of CoQ to CoQH2, which are all reversed on lowering Nai to baseline levels. Pseudo hypoxia and stabilization of HIF-1α is observed despite normal tissue oxygenation. Inhibition of mitochondrial Na/Ca-exchange with CGP-37517 or treatment with the mitochondrial ROS scavenger MitoQ prevents the metabolic alterations during Nai elevation. Elevated Nai plays a reversible role in the metabolic and functional changes and is a novel therapeutic target to correct metabolic dysfunction in heart failure.

Rise of palmitoylation: A new trick to tune NCX1 activity.

The cardiac Na+/Ca2+ Exchanger (NCX1) controls transmembrane calcium flux in numerous tissues. The only reversible post-translational modification established to regulate NCX1 is palmitoylation, which alters the ability of the exchanger to inactivate. Palmitoylation creates a binding site for the endogenous XIP domain, a region of the NCX1 intracellular loop established to inactivate NCX1. The binding site created by NCX1 palmitoylation sensitizes the transporter to XIP. Herein we summarize our recent knowledge on NCX1 palmitoylation and its association with cardiac pathologies, and discuss these findings in the light of the recent cryo-EM structures of human NCX1.

Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Cysteine post-translational modifications regulate protein interactions of caveolin-3.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.

Palmitoylation regulates the magnitude of HCN4-mediated currents in mammalian cells.

The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.

Polypharmacy in Older Adults: A Case Study.

Polypharmacy is defined as concurrent use of multiple drugs for one or more conditions. The occurrence of polypharmacy in vulnerable populations, particularly the elderly, is frequent. Increased incidents of adverse drug reactions and drug-drug interactions plus high costs are not offset by a noticeable improvement in outcome. The practice of polypharmacy persists despite frequent adverse outcomes and reduced effectiveness. We present a case in which an elderly woman presented with falls and delirium. She was taking multiple medications for anxiety and depression in addition to several psychoactive medications for pain, restless leg syndrome, muscle spasms, blood pressure and many nonpsychoactive medications for other conditions. In total, she was taking 24 medications, many of which were likely contributing to her presenting problems.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Altered Cortical Palmitoylation Induces Widespread Molecular Disturbances in Parkinson's Disease.

The relationship between Parkinson's disease (PD), the second-most common neurodegenerative disease after Alzheimer's disease, and palmitoylation, a post-translational lipid modification, is not well understood. In this study, to better understand the role of protein palmitoylation in PD and the pathways altered in this disease, we analyzed the differential palmitoyl proteome (palmitome) in the cerebral cortex of PD patients compared to controls (n = 4 per group). Data-mining of the cortical palmitome from PD patients and controls allowed us to: (i) detect a set of 150 proteins with altered palmitoylation in PD subjects in comparison with controls; (ii) describe the biological pathways and targets predicted to be altered by these palmitoylation changes; and (iii) depict the overlap between the differential palmitome identified in our study with protein interactomes of the PD-linked proteins α-synuclein, LRRK2, DJ-1, PINK1, GBA and UCHL1. In summary, we partially characterized the altered palmitome in the cortex of PD patients, which is predicted to impact cytoskeleton, mitochondrial and fibrinogen functions, as well as cell survival. Our study suggests that protein palmitoylation could have a role in the pathophysiology of PD, and that comprehensive palmitoyl-proteomics offers a powerful approach for elucidating novel cellular pathways modulated in this neurodegenerative disease.

Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies.

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

Palmitoylation regulates cellular distribution of and transmembrane Ca flux through TrpM7.

The bifunctional cation channel/kinase TrpM7 is ubiquitously expressed and regulates embryonic development and pathogenesis of several common diseases. The TrpM7 integral membrane ion channel domain regulates transmembrane movement of divalent cations, and its kinase domain controls gene expression via histone phosphorylation. Mechanisms regulating TrpM7 are elusive. It exists in two populations in the cell: at the cell surface where it controls divalent cation fluxes, and in intracellular vesicles where it controls zinc uptake and release. Here we report that TrpM7 is palmitoylated at a cluster of cysteines at the C terminal end of its Trp domain. Palmitoylation controls the exit of TrpM7 from the endoplasmic reticulum and the distribution of TrpM7 between cell surface and intracellular pools. Using the Retention Using Selective Hooks (RUSH) system, we demonstrate that palmitoylated TrpM7 traffics from the Golgi to the surface membrane whereas non-palmitoylated TrpM7 is sequestered in intracellular vesicles. We identify the Golgi-resident enzyme zDHHC17 and surface membrane-resident enzyme zDHHC5 as responsible for palmitoylating TrpM7 and find that TrpM7-mediated transmembrane calcium uptake is significantly reduced when TrpM7 is not palmitoylated. The closely related channel/kinase TrpM6 is also palmitoylated on the C terminal side of its Trp domain. Our findings demonstrate that palmitoylation controls ion channel activity of TrpM7 and that TrpM7 trafficking is dependant on its palmitoylation. We define a new mechanism for post translational modification and regulation of TrpM7 and other Trps.

Insulin-induced palmitoylation regulates the Cardiac Na+/Ca2+ exchanger NCX1.

The cardiac Na+/Ca2+ Exchanger (NCX1) controls Ca2+ extrusion from the cytosol by mediating bidirectional exchange of Na+ for Ca2+, and therefore controls cardiac relaxation. Insulin regulates Ca2+ handling in cardiac tissue through NCX1, however how insulin changes NCX1 activity is poorly understood. Palmitoylation is the only post-translational modification identified to alter NCX1 activity. Here we show that insulin triggers local structural re-arrangements within existing NCX1 dimers by inducing their palmitoylation, thus tunes NCX1 inactivation through a zDHHC5-dependent mechanism in multiple cell types. By activating fatty acid and fatty acyl CoA synthesis insulin promotes palmitoylation of the zDHHC5 active site, which leads to enhanced NCX1 palmitoylation. Our findings represent a new mechanism to regulate the palmitoylation of numerous zDHHC5 substrates.

Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

Protein S-Palmitoylation: advances and challenges in studying a therapeutically important lipid modification.

The lipid post-translational modification S-palmitoylation is a vast developing field, with the modification itself and the enzymes that catalyse the reversible reaction implicated in a number of diseases. In this review, we discuss the past and recent advances in the experimental tools used in this field, including pharmacological tools, animal models and techniques to understand how palmitoylation controls protein localisation and function. Additionally, we discuss the obstacles to overcome in order to advance the field, particularly to the point at which modulating palmitoylation may be achieved as a therapeutic strategy.

Identifying the beta-site amyloid precursor protein cleaving enzyme 1 interactome through the proximity-dependent biotin identification assay.

Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a key drug target against Alzheimer's Disease however, due to its promiscuous proteolytic activity, little is known about its physiological functions. Previous studies have analysed BACE1 cleavage products to examine BACE1 interactions and determine substrates, but these studies cannot establish non-enzymatic (and potentially functional) associations. This study used the biotin identification proximity assay to establish the BACE1 interactome in healthy neuronal cells and identified interactions involved in BACE1 trafficking, post-translational modification and substrates. Furthermore, this method has identified a putative novel role for BACE1 in sex hormone signalling and haem regulation through interaction with the progesterone receptor membrane component 2 (PGRC2). Data are available via ProteomeXchange with identifier PXD021464.