Regulatory mechanisms controlling store-operated calcium entry.

Calcium influx through plasma membrane ion channels is crucial for many events in cellular physiology. Cell surface stimuli lead to the production of inositol 1,4,5-trisphosphate (IP3), which binds to IP3 receptors (IP3R) in the endoplasmic reticulum (ER) to release calcium pools from the ER lumen. This leads to the depletion of ER calcium pools, which has been termed store depletion. Store depletion leads to the dissociation of calcium ions from the EF-hand motif of the ER calcium sensor Stromal Interaction Molecule 1 (STIM1). This leads to a conformational change in STIM1, which helps it to interact with the plasma membrane (PM) at ER:PM junctions. At these ER:PM junctions, STIM1 binds to and activates a calcium channel known as Orai1 to form calcium-release activated calcium (CRAC) channels. Activation of Orai1 leads to calcium influx, known as store-operated calcium entry (SOCE). In addition to Orai1 and STIM1, the homologs of Orai1 and STIM1, such as Orai2/3 and STIM2, also play a crucial role in calcium homeostasis. The influx of calcium through the Orai channel activates a calcium current that has been termed the CRAC current. CRAC channels form multimers and cluster together in large macromolecular assemblies termed "puncta". How CRAC channels form puncta has been contentious since their discovery. In this review, we will outline the history of SOCE, the molecular players involved in this process, as well as the models that have been proposed to explain this critical mechanism in cellular physiology.

Regulatory mechanisms controlling store-operated calcium entry.

Calcium influx through plasma membrane ion channels is crucial for many events in cellular physiology. Cell surface stimuli lead to the production of inositol 1,4,5-trisphosphate (IP3), which binds to IP3 receptors (IP3R) in the endoplasmic reticulum (ER) to release calcium pools from the ER lumen. This leads to the depletion of ER calcium pools, which has been termed store depletion. Store depletion leads to the dissociation of calcium ions from the EF-hand motif of the ER calcium sensor Stromal Interaction Molecule 1 (STIM1). This leads to a conformational change in STIM1, which helps it to interact with the plasma membrane (PM) at ER:PM junctions. At these ER:PM junctions, STIM1 binds to and activates a calcium channel known as Orai1 to form calcium release-activated calcium (CRAC) channels. Activation of Orai1 leads to calcium influx, known as store-operated calcium entry (SOCE). In addition to Orai1 and STIM1, the homologs of Orai1 and STIM1, such as Orai2/3 and STIM2, also play a crucial role in calcium homeostasis. The influx of calcium through the Orai channel activates a calcium current that has been termed the CRAC current. CRAC channels form multimers and cluster together in large macromolecular assemblies termed "puncta". How CRAC channels form puncta has been contentious since their discovery. In this review, we will outline the history of SOCE, the molecular players involved in this process, as well as the models that have been proposed to explain this critical mechanism in cellular physiology.

Regulation of T cell function by protein S-acylation.

S-acylation, the reversible lipidation of free cysteine residues with long-chain fatty acids, is a highly dynamic post-translational protein modification that has recently emerged as an important regulator of the T cell function. The reversible nature of S-acylation sets this modification apart from other forms of protein lipidation and allows it to play a unique role in intracellular signal transduction. In recent years, a significant number of T cell proteins, including receptors, enzymes, ion channels, and adaptor proteins, were identified as S-acylated. It has been shown that S-acylation critically contributes to their function by regulating protein localization, stability and protein-protein interactions. Furthermore, it has been demonstrated that zDHHC protein acyltransferases, the family of enzymes mediating this modification, also play a prominent role in T cell activation and differentiation. In this review, we aim to highlight the diversity of proteins undergoing S-acylation in T cells, elucidate the mechanisms by which reversible lipidation can impact protein function, and introduce protein acyltransferases as a novel class of regulatory T cell proteins.

The Prevalence of Viral Pathogens among Bats in Kazakhstan.

Bats carry thousands of viruses from 28 different families. To determine the presence of various pathogens in bat populations in Kazakhstan, 1149 samples (393 oropharyngeal swabs, 349 brain samples, 407 guano) were collected. The samples were collected from four species of bats (Vespertilio murinus, Nyctalus noctula, Myotis blythii, Eptesicus serotinus) in nine regions. The Coronavirus RNA was found in 38 (4.75%) samples, and the rabies virus in 27 (7.74%) samples from bats. Coronaviruses and the rabies virus were found in bats in six out of nine studied areas. The RNAs of SARS-CoV-2, MERS, TBE, CCHF, WNF, influenza A viruses were not detected in the bat samples. The phylogeny of the RdRp gene of 12 samples made it possible to classify them as alphacoronaviruses and divide them into two groups. The main group (n = 11) was closely related to bat coronaviruses from Ghana, Zimbabwe and Kenya. The second group (n = 1) was closely related to viruses previously isolated in the south of Kazakhstan. The phylogeny of the N gene sequence from a bat from west Kazakhstan revealed its close relationship with isolates from the Cosmopolitan group of rabies viruses (Central Asia). These results highlight the need for a continuous monitoring of volatile populations to improve the surveillance and detection of infectious diseases.

Dynamic S-acylation of the ER-resident protein stromal interaction molecule 1 (STIM1) is required for store-operated Ca2+ entry.

Many cell surface stimuli cause calcium release from endoplasmic reticulum (ER) stores to regulate cellular physiology. Upon ER calcium store depletion, the ER-resident protein stromal interaction molecule 1 (STIM1) physically interacts with plasma membrane protein Orai1 to induce calcium release-activated calcium (CRAC) currents that conduct calcium influx from the extracellular milieu. Although the physiological relevance of this process is well established, the mechanism supporting the assembly of these proteins is incompletely understood. Earlier we demonstrated a previously unknown post-translational modification of Orai1 with long-chain fatty acids, known as S-acylation. We found that S-acylation of Orai1 is dynamically regulated in a stimulus-dependent manner and essential for its function as a calcium channel. Here using the acyl resin-assisted capture assay, we show that STIM1 is also rapidly S-acylated at cysteine 437 upon ER calcium store depletion. Using a combination of live cell imaging and electrophysiology approaches with a mutant STIM1 protein, which could not be S-acylated, we determined that the S-acylation of STIM1 is required for the assembly of STIM1 into puncta with Orai1 and full CRAC channel function. Together with the S-acylation of Orai1, our data suggest that stimulus-dependent S-acylation of CRAC channel components Orai1 and STIM1 is a critical mechanism facilitating the CRAC channel assembly and function.

S-acylation of Orai1 regulates store-operated Ca2+ entry.

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.

Ca2+-dependent protein acyltransferase DHHC21 controls activation of CD4+ T cells.

Despite the recognized significance of reversible protein lipidation (S-acylation) for T cell receptor signal transduction, the enzymatic control of this post-translational modification in T cells remains poorly understood. Here, we demonstrate that DHHC21 (also known as ZDHHC21), a member of the DHHC family of mammalian protein acyltransferases, mediates T cell receptor-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21dep mice, which express a functionally deficient version of DHHC21, we show that DHHC21 is a Ca2+/calmodulin-dependent enzyme critical for activation of naïve CD4+ T cells in response to T cell receptor stimulation. We find that disruption of the Ca2+/calmodulin-binding domain of DHHC21 does not affect thymic T cell development but prevents differentiation of peripheral CD4+ T cells into Th1, Th2 and Th17 effector T helper lineages. Our findings identify DHHC21 as an essential component of the T cell receptor signaling machinery and define a new role for protein acyltransferases in regulation of T cell-mediated immunity.

Simvastatin modulates estrogen signaling in uterine leiomyoma via regulating receptor palmitoylation, trafficking and degradation.

Uterine leiomyomas or fibroids are the most common tumors of the female reproductive tract. Estrogen (E2), a steroid-derived hormone, and its receptors (ERs), particularly ER-α, are important drivers for the development and growth of leiomyomas. We previously demonstrated that simvastatin, a drug used for hyperlipidemia, also possesses anti-leiomyoma properties. The aim of this work is to investigate the impact of simvastatin on ER-α signaling in leiomyoma cells, including its expression, downstream signaling, transcriptional activity, post-translational modification, trafficking and degradation. Primary and immortalized human uterine leiomyoma (HuLM) cells were used for in vitro experiments. Immunodeficient mice xenografted with human leiomyoma tissue explants were used for in vivo studies. Leiomyoma samples were obtained from patients enrolled in an ongoing double-blinded, phase II, randomized controlled trial. Here, we found that simvastatin significantly reduced E2-induced proliferation and PCNA expression. In addition, simvastatin reduced total ER-α expression in leiomyoma cells and altered its subcellular localization by inhibiting its trafficking to the plasma membrane and nucleus. Simvastatin also inhibited E2 downstream signaling, including ERK and AKT pathways, E2/ER transcriptional activity and E2-responsive genes. To explain simvastatin effects on ER-α level and trafficking, we examined its effects on ER-α post-translational processing. We noticed that simvastatin reduced ER-α palmitoylation; a required modification for its stability, trafficking to plasma membrane, and signaling. We also observed an increase in ubiquitin-mediated ER-α degradation. Importantly, we found that the effects of simvastatin on ER-α expression were recapitulated in the xenograft leiomyoma mouse model and human tissues. Thus, our data suggest that simvastatin modulates several E2/ER signaling targets with potential implications in leiomyoma therapy and beyond.

Fatty acid mobilization from adipose tissue is mediated by CD36 posttranslational modifications and intracellular trafficking.

The mechanism controlling long-chain fatty acid (LCFA) mobilization from adipose tissue is not well understood. Here, we investigated how the LCFA transporter CD36 regulates this process. By using tissue-specific KO mouse models, we showed that CD36 in adipocytes and endothelial cells mediated both LCFA deposition into and release from adipose tissue. We demonstrated the role of adipocytic and endothelial CD36 in promoting tumor growth and chemoresistance conferred by adipose tissue-derived LCFAs. We showed that dynamic cysteine S-acylation of CD36 in adipocytes, endothelial cells, and cancer cells mediated intercellular LCFA transport. We demonstrated that lipolysis induction in adipocytes triggered CD36 deacylation and deglycosylation, as well as its dissociation from interacting proteins, prohibitin-1 (PHB) and annexin 2 (ANX2). Our data indicate that lipolysis triggers caveolar endocytosis and translocation of CD36 from the cell membrane to lipid droplets. This study suggests a mechanism for both outside-in and inside-out cellular LCFA transport regulated by CD36 S-acylation and its interactions with PHB and ANX2.

T cell receptor-dependent S-acylation of ZAP-70 controls activation of T cells.

ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity.

Pre-Transplant Marital Status and Hematopoietic Cell Transplantation Outcomes

Background: Evidence about the impact of marital status before hematopoietic cell transplantation (hct) on outcomes after hct is conflicting. Methods: We identified patients 40 years of age and older within the Center for International Blood and Marrow Transplant Research registry who underwent hct between January 2008 and December 2015. Marital status before hct was declared as one of: married or living with a partner, single (never married), separated or divorced, and widowed. We performed a multivariable analysis to determine the association of marital status with outcomes after hct. Results: We identified 10,226 allogeneic and 5714 autologous hct cases with, respectively, a median follow-up of 37 months (range: 1-102 months) and 40 months (range: 1-106 months). No association between marital status and overall survival was observed in either the allogeneic (p = 0.58) or autologous (p = 0.17) setting. However, marital status was associated with grades 2-4 acute graft-versus-host disease (gvhd), p < 0.001, and chronic gvhd, p = 0.04. The risk of grades 2-4 acute gvhd was increased in separated compared with married patients [hazard ratio (hr): 1.13; 95% confidence interval (ci): 1.03 to 1.24], and single patients had a reduced risk of grades 2-4 acute gvhd (hr: 0.87; 95% ci: 0.77 to 0.98). The risk of chronic gvhd was lower in widowed compared with married patients (hr: 0.82; 95% ci: 0.67 to 0.99). Conclusions: Overall survival after hct is not influenced by marital status, but associations were evident between marital status and grades 2-4 acute and chronic gvhd. To better appreciate the effects of marital status and social support, future research should consider using validated scales to measure social support and patient and caregiver reports of caregiver commitment, and to assess health-related quality of life together with health care utilization.

Activity Dependent Inhibition of AMPA Receptors by Zn2.

Zn2+ has been shown to have a wide range of modulatory effects on neuronal AMPARs. However, the mechanism of modulation is largely unknown. Here we show that Zn2+ inhibits GluA2(Q) homomeric receptors in an activity- and voltage-dependent manner, indicating a pore block mechanism. The rate of inhibition is slow, in the hundreds of milliseconds at millimolar Zn2+ concentrations; hence, the inhibition is only observed in the residual nondesensitizing currents. Consequently, the inhibition is higher for GluA2 receptors in complex with auxiliary subunits γ2 and γ8 where the residual activation is larger. The extent of inhibition is also dependent on charge at site 607, the site that undergoes RNA editing in GluA2 subunits replacing glutamine to arginine, with the percent inhibition being lower and IC50 being higher for the edited GluA2(R) relative to unedited GluA2(Q) and to GluA2(Q607E), a mutation observed in the genetic screen of a patient exhibiting developmental delays. We also show that Zn2+ inhibition is significant during rapid repetitive activity with pulses of millimolar concentrations of glutamate in both receptors expressed in HEK cells as well as in native receptors in cortical neurons of C57BL/6J mice of either sex, indicating a physiological relevance of this inhibition.SIGNIFICANCE STATEMENT Zn2+ is present along with glutamate in synaptic vesicles and coreleased during synaptic transmission, modulating the postsynaptic ionotropic glutamate receptors. While Zn2+ inhibition of the NMDA subtype of the ionotropic glutamate receptors is well characterized, the mechanism of modulation of the AMPA subtype is much less known. Here we have systematically studied Zn2+ inhibition of AMPARs by varying calcium permeability, auxiliary subunits, and activation levels and show that Zn2+ inhibits AMPARs in an activity-dependent manner, opening up this pathway as a means to pharmacologically modulate the receptors.

Regulation of T cell receptor signaling by protein acyltransferase DHHC21.

S-acylation reversible-post-translational lipidation of cysteine residues-is emerging as an important regulatory mechanism in T cell signaling. Dynamic S-acylation is critical for protein recruitment into the T cell receptor complex and initiation of the subsequent signaling cascade. However, the enzymatic control of protein S-acylation in T cells remains poorly understood. Here, we report a previously uncharacterized role of DHHC21, a member of the mammalian family of DHHC protein acyltransferases, in regulation of the T cell receptor pathway. We found that loss of DHHC21 prevented S-acylation of key T cell signaling proteins, resulting in disruption of the early signaling events and suppressed expression of T cell activation markers. Furthermore, downregulation of DHHC21 prevented activation and differentiation of naïve T cells into effector subtypes. Together, our study provides the first direct evidence that DHHC protein acyltransferases can play an essential role in regulation of T cell-mediated immunity.

Third window approach assisted middle meatal antrostomy: prospective cohort study of the two ports technique in management of hard to reach maxillary sinus pathology.

OBJECTIVE: This study aimed to assess the outcomes of a prelacrimal recess approach assisted middle meatal antrostomy in the management of hard to reach maxillary sinus pathologies. METHOD: Twenty-five patients with maxillary sinus pathology underwent prelacrimal recess approach assisted middle meatal antrostomy (with a prelacrimal recess width of more than 3 mm). Patients were prospectively evaluated using both the Arabic version of the Sino-Nasal Outcome Test-22 and nasal endoscopy at least 6 months post-operatively. RESULTS: Our study included 25 maxillary sinuses (13 with antrochoanal polyps, 10 with maxillary fungal ball and 2 with a migrated part of a tooth). At a mean follow-up period of 10.9 months, all patients showed significant improvement in total mean Sino-Nasal Outcome Test-22 score. There was recurrence of one case with antrochoanal polyp and two cases with asymptomatic synechia. Injury to the nasolacrimal duct was not reported. CONCLUSION: A prelacrimal recess approach assisted middle meatal antrostomy is a reliable and safe technique to manage pathologies in hard to reach regions within the maxillary sinus.

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture.

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

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.

Aggregated and Hyperstable Damage-Associated Molecular Patterns Are Released During ER Stress to Modulate Immune Function.

Chronic ER stress occurs when protein misfolding in the Endoplasmic reticulum (ER) lumen remains unresolved despite activation of the unfolded protein response. We have shown that traumatic injury such as a severe burn leads to chronic ER stress in vivo leading to systemic inflammation which can last for more than a year. The mechanisms linking chronic ER stress to systemic inflammatory responses are not clear. Here we show that induction of chronic ER stress leads to the release of known and novel damage-associated molecular patterns (DAMPs). The secreted DAMPs are aggregated and markedly protease resistant. ER stress-derived DAMPs activate dendritic cells (DCs) which are then capable of polarizing naïve T cells. Our findings indicate that induction of chronic ER stress may lead to the release of hyperstable DAMPs into the circulation resulting in persistent systemic inflammation and adverse outcomes.

Antimicrobial Efficacy of Different Natural Extracts Against Persistent Root Canal Pathogens: An In vitro Study.

INTRODUCTION: The spread of drug-resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. Extracts of plants such as flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, and roots have evoked interest as sources of natural products. Irrigation with a broad-spectrum antiseptic substance and inter-appointment intracanal medication has become a standard regimen in root canal therapy. AIM: The aim of this study is to compare the antimicrobial efficacy of different natural extracts such as guava leaf extract, Aloe vera extract, papaya leaf extract, and cashew apple extract against Enterococcus faecalis and Candida albicans. MATERIALS AND METHODS: The antimicrobial activity was determined using agar diffusion test. The solutions were divided into four groups: Group I - guava leaf extract, Group II - A. vera extract, and Group III - papaya leaf extract, and Group IV - cashew apple extract. The zones of inhibition of growth were recorded. The strains used for this study were E. faecalis ATCC 29212 and C. albicans ATCC 90028. RESULTS AND CONCLUSION: Sodium hypochlorite had demonstrated the best results among the tested solutions. Among the herbal extracts, cashew apple extract and guava leaf extract had shown statistically significant activity against E. faecalis and C. albicans.

Genetic diversity of avian avulavirus 1 (Newcastle disease virus genotypes VIg and VIIb) circulating in wild birds in Kazakhstan.

In order to improve current understanding of the molecular epidemiology of avian avulavirus 1 (AAvV-1, formerly avian paramyxovirus 1) in wild birds in Kazakhstan, 860 cloacal swab samples were evaluated. Samples were collected from 37 families of wild birds in nine different regions in the years 2011 and 2014. Overall, 54 positive samples (4.2%) were detected from 17 different families of wild birds, and 16 AAvV-1 isolates were characterized. Three of the isolates contained the fusion protein cleavage site motif RRQKR, and 13 contained KRQKR, which is typical for pathogenic strains of AAvV-1. The AAvV-1 isolates were found to belong to the genotypes VIg and VIIb.

Stentless mirrored L-shaped septonasal flap versus stented flapless technique for endoscopic endonasal repair of bilateral congenital choanal atresia: a prospective randomised controlled study.

OBJECTIVES: To compare the outcomes of endoscopic repair of bilateral congenital choanal atresia using a flap technique without stenting versus endoscopic repair using stenting without a flap. METHODS: A prospective randomised controlled study was conducted, comprising 72 patients with bilateral congenital choanal atresia. The patients were randomised into two groups. Group A (42 patients) underwent endoscopic repair using a mirrored L-shaped flap without stenting, and group B (30 patients) underwent endoscopic repair using stenting without a flap. RESULTS: At a mean follow-up period of 18.2 months, endoscopic assessment revealed a patent posterior choana in 81 per cent and 83.33 per cent of patients in group A and group B respectively. Choanal stenosis occurred in 21.40 per cent and 33.33 per cent of patients in group A and group B respectively. Granulation tissue was observed in 28.6 per cent and 53.3 per cent of patients in group A and group B respectively. CONCLUSION: The endoscopic approach utilising a flap without stenting is safe and effective, with a high success rate.