Regulation of Dynamic Protein S-Acylation.

Protein S-acylation is the reversible addition of fatty acids to the cysteine residues of target proteins. It regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interactions, protein stability, and protein conformation. This process is regulated by palmitoyl acyltransferases that have the conserved amino acid sequence DHHC at their active site. Although they have conserved catalytic cores, DHHC enzymes vary in their protein substrate selection, lipid substrate preference, and regulatory mechanisms. Alterations in DHHC enzyme function are associated with many human diseases, including cancers and neurological conditions. The removal of fatty acids from acylated cysteine residues is catalyzed by acyl protein thioesterases. Notably, S-acylation is now known to be a highly dynamic process, and plays crucial roles in signaling transduction in various cell types. In this review, we will explore the recent findings on protein S-acylation, the enzymatic regulation of this process, and discuss examples of dynamic S-acylation.

DHHC5 Mediates ß-Adrenergic Signaling in Cardiomyocytes by Targeting Gα Proteins.

S-palmitoylation is a reversible posttranslational modification that plays an important role in regulating protein localization, trafficking, and stability. Recent studies have shown that some proteins undergo extremely rapid palmitoylation/depalmitoylation cycles after cellular stimulation supporting a direct signaling role for this posttranslational modification. Here, we investigated whether ß-adrenergic stimulation of cardiomyocytes led to stimulus-dependent palmitoylation of downstream signaling proteins. We found that ß-adrenergic stimulation led to rapidly increased Gαs and Gαi palmitoylation. The kinetics of palmitoylation was temporally consistent with the downstream production of cAMP and contractile responses. We identified the plasma membrane-localized palmitoyl acyltransferase DHHC5 as an important mediator of the stimulus-dependent palmitoylation in cardiomyocytes. Knockdown of DHHC5 showed that this enzyme is necessary for palmitoylation of Gαs, Gαi, and functional responses downstream of ß-adrenergic stimulation. A palmitoylation assay with purified components revealed that Gαs and Gαi are direct substrates of DHHC5. Finally, we provided evidence that the C-terminal tail of DHHC5 can be palmitoylated in response to stimulation and such modification is important for its dynamic localization and function in the plasma membrane. Our results reveal that DHHC5 is a central regulator of signaling downstream of ß-adrenergic receptors in cardiomyocytes.

Functionally redundant control of cardiac hypertrophic signaling by inositol 1,4,5-trisphosphate receptors.

Calcium plays an integral role to many cellular processes including contraction, energy metabolism, gene expression, and cell death. The inositol 1, 4, 5-trisphosphate receptor (IP3R) is a calcium channel expressed in cardiac tissue. There are three IP3R isoforms encoded by separate genes. In the heart, the IP3R-2 isoform is reported to being most predominant with regards to expression levels and functional significance. The functional roles of IP3R-1 and IP3R-3 in the heart are essentially unexplored despite measureable expression levels. Here we show that all three IP3Rs isoforms are expressed in both neonatal and adult rat ventricular cardiomyocytes, and in human heart tissue. The three IP3R proteins are expressed throughout the cardiomyocyte sarcoplasmic reticulum. Using isoform specific siRNA, we found that expression of all three IP3R isoforms are required for hypertrophic signaling downstream of endothelin-1 stimulation. Mechanistically, IP3Rs specifically contribute to activation of the hypertrophic program by mediating the positive inotropic effects of endothelin-1 and leading to downstream activation of nuclear factor of activated T-cells. Our findings highlight previously unidentified functions for IP3R isoforms in the heart with specific implications for hypertrophic signaling in animal models and in human disease.

Protein Lipidation As a Regulator of Apoptotic Calcium Release: Relevance to Cancer.

Calcium is a critical regulator of cell death pathways. One of the most proximal events leading to cell death is activation of plasma membrane and endoplasmic reticulum-resident calcium channels. A large body of evidence indicates that defects in this pathway contribute to cancer development. Although we have a thorough understanding of how downstream elevations in cytosolic and mitochondrial calcium contribute to cell death, it is much less clear how calcium channels are activated upstream of the apoptotic stimulus. Recently, it has been shown that protein lipidation is a potent regulator of apoptotic signaling. Although classically thought of as a static modification, rapid and reversible protein acylation has emerged as a new signaling paradigm relevant to many pathways, including calcium release and cell death. In this review, we will discuss the role of protein lipidation in regulating apoptotic calcium signaling with direct therapeutic relevance to cancer.

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis.

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.

A Transport Model for Estimating the Time Course of ERK Activation in the C. elegans Germline.

The Caenorhabditis elegans germline is a well-studied model system for investigating the control of cell fate by signaling pathways. Cell signals at the distal tip of the germline promote cell proliferation; just before the loop, signals couple cell maturation to organism-level nutrient status; at the proximal end of the germline, signals coordinate oocyte maturation and fertilization in the presence of sperm. The latter two events require dual phosphorylation and activation of ERK, the effector molecule of the Ras/MAPK cascade. In C. elegans, ERK is known as MPK-1. At this point, none of today's methods for real-time monitoring of dually phosphorylated MPK-1 are working in the germline. Consequently, quantitative understanding of the MPK-1-dependent processes during germline development is limited. Here, we make a step toward advancing this understanding using a model-based framework that reconstructs the time course of MPK-1 activation from a snapshot of a fixed germline. Our approach builds on a number of recent studies for estimating temporal dynamics from fixed organisms, but takes advantage of the anatomy of the germline to simplify the analysis. Our model predicts that the MPK-1 signal turns on ∼30 h into germ cell progression and peaks ∼7 h later.

The Effect of Health Literacy on Medication Knowledge and Medication Discrepancy in Chinese Americans.

Background. Functional health literacy (FHL) is increasingly recognized as a useful predictor of health outcomes in different populations. However, the effect of FHL on medication knowledge and medication discrepancy in Chinese Americans is not well defined. Objectives. To examine the effects of FHL on medication knowledge and medication discrepancy in Chinese American patients. Methods. This was a cross-sectional study conducted at an academic internal medicine clinic. The Short Test of Functional Health Literacy in Adults was used to assess participants' FHL. Data for patients' demographic information, medication knowledge, and medication discrepancy (direction discrepancy and name discrepancy) were collected through patient interviews and chart reviews. The primary outcome was medication knowledge of purpose and the secondary outcomes included medication direction discrepancy and medication name discrepancy. Results. Of the 158 Chinese American patients who participated in the study, 54% had adequate FHL. More participants with adequate FHL had correct medication knowledge compared to participants with inadequate FHL (87% vs 56%, respectively, odds ratio = 3.4, 95% confidence interval = 1.2-9.7). Fewer participants with adequate FHL had medication direction discrepancy compared to those with inadequate FHL (42% vs 62%, odds ratio = 0.18, 95% confidence interval = 0.06-0.55). Both adequate and inadequate FHL groups had high prevalence of medication name discrepancy (77% vs 89%) even though the between-group difference was insignificant. Conclusions. Adequate FHL among Chinese American patients is significantly associated with increased medication knowledge of purpose and decreased medication direction discrepancy. Both adequate and inadequate FHL groups had high prevalence of medication name discrepancy.