RFCM-PALM: In-Silico Prediction of S-Palmitoylation Sites in the Synaptic Proteins for Male/Female Mouse Data.

S-palmitoylation is a reversible covalent post-translational modification of cysteine thiol side chain by palmitic acid. S-palmitoylation plays a critical role in a variety of biological processes and is engaged in several human diseases. Therefore, identifying specific sites of this modification is crucial for understanding their functional consequences in physiology and pathology. We present a random forest (RF) classifier-based consensus strategy (RFCM-PALM) for predicting the palmitoylated cysteine sites on synaptic proteins from male/female mouse data. To design the prediction model, we have introduced a heuristic strategy for selection of the optimum set of physicochemical features from the AAIndex dataset using (a) K-Best (KB) features, (b) genetic algorithm (GA), and (c) a union (UN) of KB and GA based features. Furthermore, decisions from best-trained models of the KB, GA, and UN-based classifiers are combined by designing a three-star quality consensus strategy to further refine and enhance the scores of the individual models. The experiment is carried out on three categorized synaptic protein datasets of a male mouse, female mouse, and combined (male + female), whereas in each group, weighted data is used as training, and knock-out is used as the hold-out set for performance evaluation and comparison. RFCM-PALM shows ~80% area under curve (AUC) score in all three categories of datasets and achieve 10% average accuracy (male-15%, female-15%, and combined-7%) improvements on the hold-out set compared to the state-of-the-art approaches. To summarize, our method with efficient feature selection and novel consensus strategy shows significant performance gains in the prediction of S-palmitoylation sites in mouse datasets.

S-Palmitoylation of Synaptic Proteins as a Novel Mechanism Underlying Sex-Dependent Differences in Neuronal Plasticity.

Although sex differences in the brain are prevalent, the knowledge about mechanisms underlying sex-related effects on normal and pathological brain functioning is rather poor. It is known that female and male brains differ in size and connectivity. Moreover, those differences are related to neuronal morphology, synaptic plasticity, and molecular signaling pathways. Among different processes assuring proper synapse functions are posttranslational modifications, and among them, S-palmitoylation (S-PALM) emerges as a crucial mechanism regulating synaptic integrity. Protein S-PALM is governed by a family of palmitoyl acyltransferases, also known as DHHC proteins. Here we focused on the sex-related functional importance of DHHC7 acyltransferase because of its S-PALM action over different synaptic proteins as well as sex steroid receptors. Using the mass spectrometry-based PANIMoni method, we identified sex-dependent differences in the S-PALM of synaptic proteins potentially involved in the regulation of membrane excitability and synaptic transmission as well as in the signaling of proteins involved in the structural plasticity of dendritic spines. To determine a mechanistic source for obtained sex-dependent changes in protein S-PALM, we analyzed synaptoneurosomes isolated from DHHC7-/- (DHHC7KO) female and male mice. Our data showed sex-dependent action of DHHC7 acyltransferase. Furthermore, we revealed that different S-PALM proteins control the same biological processes in male and female synapses.

Stress-induced Changes in the S-palmitoylation and S-nitrosylation of Synaptic Proteins.

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by crosstalk with other posttranslational modifications, such as S-nitrosylation. S-nitrosylation is a covalent modification of cysteine thiol by nitric oxide and can modulate protein functions. Therefore, simultaneous identification of endogenous site-specific proteomes of both cysteine modifications under certain biological conditions offers new insights into the regulation of functional pathways. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach Palmitoylation And Nitrosylation Interplay Monitoring (PANIMoni), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteine in a mouse model of chronic stress. Among a total of 813 S-PALM and 620 S-NO cysteine sites that were characterized on 465 and 360 proteins, respectively, we sought to identify those that were differentially affected by stress. Our data show involvement of S-palmitoylation and S-nitrosylation crosstalk in the regulation of 122 proteins including receptors, scaffolding proteins, regulatory proteins and cytoskeletal components. Our results suggest that atypical crosstalk between the S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity might be one of the main events associated with chronic stress disorder, leading to destabilization in synaptic networks.

Insights Into Protein S-Palmitoylation in Synaptic Plasticity and Neurological Disorders: Potential and Limitations of Methods for Detection and Analysis.

S-palmitoylation (S-PALM) is a lipid modification that involves the linkage of a fatty acid chain to cysteine residues of the substrate protein. This common posttranslational modification (PTM) is unique among other lipid modifications because of its reversibility. Hence, like phosphorylation or ubiquitination, it can act as a switch that modulates various important physiological pathways within the cell. Numerous studies revealed that S-PALM plays a crucial role in protein trafficking and function throughout the nervous system. Notably, the dynamic turnover of palmitate on proteins at the synapse may provide a key mechanism for rapidly changing synaptic strength. Indeed, palmitate cycling on postsynaptic density-95 (PSD-95), the major postsynaptic density protein at excitatory synapses, regulates the number of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and thus affects synaptic transmission. Accumulating evidence suggests a relationship between impairments in S-PALM and severe neurological disorders. Therefore, determining the precise levels of S-PALM may be essential for understanding the ways in which this PTM is regulated in the brain and controls synaptic dynamics. Protein S-PALM can be characterized using metabolic labeling methods and biochemical tools. Both approaches are discussed herein in the context of specific methods and their advantages and disadvantages. This review clearly shows progress in the field, which has led to the development of new, more sensitive techniques that enable the detection of palmitoylated proteins and allow predictions of potential palmitate binding sites. Unfortunately, one significant limitation of these approaches continues to be the inability to use them in living cells.

A Simple Method for TPMT and ITPA Genotyping Using Multiplex HRMA for Patients Treated with Thiopurine Drugs.

Thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA) are crucial enzymes involved in the metabolism of thiopurine drugs: azathioprine and 6-mercaptopurine, used in the treatment of leukemia or inflammatory bowel diseases (IBD). The activity in these enzymes correlates with the genetic polymorphism of the TPMT and ITPA genes, respectively, which determines an individual reaction and dosing of thiopurines. Three main TPMT alleles: TPMT*2 (c.238G>C), TPMT*3A (c.460G>A, c.719A>G) and TPMT*3C (c.719A>G) account for 80-95 % of inherited TPMT deficiency in different populations in the world. In the ITPA gene, a c.94C>A mutation is significantly associated with an adverse thiopurine reaction. The aim of this study was to develop a quick and highly sensitive method for determining major TPMT and ITPA alleles. Here we present the molecular test for genotyping c.238G>C, c.460G>A, c.719A>G and c.94C>A changes based on multiplex high resolution melting analysis (HRMA). We analyzed DNA samples from 100 clinically diagnosed IBD patients treated with thiopurine drugs, and a known genotype in the positions 238, 460 and 719 of the TPMT gene as well as in position 94 of the ITPA gene. Our results obtained with multiplex HRMA indicated 100 % accuracy in comparison with data from restriction fragments length polymorphism (RFLP) and standard DNA sequencing. We conclude, that multiplex HRMA can be used as a quick, sensitive and efficient alternative diagnostic method compared to conventional techniques for the determination of TPMT*2, TPMT*3A and TPMT*3C alleles and c.94C>A change in the ITPA gene.