30 research outputs found
Calmodulin Interacts and Regulates Enzyme Activity of the Mammalian Sperm Phospholipase C
Sperm-specific Phospholipase C zeta (PLCĪ¶) is widely considered to be the sole, physiological stimulus responsible for the generation of Ca2+ oscillations that induce egg activation and early embryo development during mammalian fertilization. PLCĪ¶, which is delivered from the fertilizing sperm into the egg cytoplasm, catalyzes the hydrolysis of its membrane-bound phospholipid substrate phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], triggering the cytoplasmic Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. Despite the recent advances the detailed regulatory mechanism of PLCĪ¶ is still unclear, as binding partners of this protein within the sperm or the fertilizing egg have not yet been identified. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotic cells. A previous study has reported that CaM directly interacts and regulates the activity of PLC delta 1 protein, a somatic PLC isoform with structural similarities to sperm PLCĪ¶. Bioinformatics analysis revealed putative CaM-binding sites on PLCĪ¶ sequence. In the present study, we have used co-immunoprecipitation analysis and we show that in the presence of Ca2+, human PLCĪ¶ directly interacts with CaM. Isothermal titration calorimetry (ITC) experiments were performed to map the interaction. Three different peptides corresponding to disparate sequences within human PLCĪ¶ were used and it was shown that PLCĪ¶ interacts with CaM via one region of the molecule. In addition, recombinant proteins corresponding to the N- and C-lobe of human CaM were used for ITC experiments, which revealed that CaM interacts with PLCĪ¶ in the presence of Ca2+, only through one of its lobe domains. In vitro PIP2 hydrolysis assays revealed that CaM alters PLCĪ¶ PIP2 hydrolytic activity at high Ca2+ concentrations and, as suggested by liposome binding assays, this appears to be due to CaM binding to PLCĪ¶ affecting proper access of the enzyme active site to its substrate PI(4,5)P2
Defective Interaction of Cam with RyR2 Cam-Binding Pocket Might Contribute to Arrhythmogenic Cardiac Disease
Ryanodine receptor 2 (RyR2) is a large transmembrane calcium (Ca2+) release channel that mediates Ca2 release from the sarcoplasmic reticulum to activate cardiac muscle contraction. Calmodulin (CaM) regulation of RyR2 is essential for normal cardiac function. A number of linear fragments of RyR2 have been reported as potential CaM-binding sequences. The sequence 3583-3603aa of human RyR2, which is highly conserved among mammalian isoforms, has been identified as a CaM-binding site in almost all relevant studies and therefore this region is considered as a well-established CaM-binding domain of RyRs. Besides 3583-3603aa region, other RyR2 regions have been also reported as potential CaM-binding sequences. Herein, we used recombinant wild-type CaMprotein and isothermal titration calorimetry (ITC) experiments to screen a number of RyR2-specific synthetic peptides corresponding to the region 4240-4277aa of RyR2, which has been previously proposed as a putative CaM-binding RyR2 region. From all the synthetic peptides screened, a peptide corresponding to 4255-4271aa region of human RyR2 was found to interact with significant affinity with RyR2, in the presence and absence of Ca2+ (Kd values 0.60 and 16.58 Ī¼M, respectively). Moreover, investigation of the interaction of four arrhythmogenic CaM mutants (N98I, D132E, D134H and Q136P) with this synthetic peptide, as well as the peptide corresponding to the well-established CaM-binding domain of RyR2 (3583-3603aa), revealed that all mutants show disparate binding properties to these two RyR2 peptides, which have been previously proposed to contribute to a putative intra-subunit CaM-binding pocket. Our findings extend our previous observations suggesting that CaM mutations may trigger arrhythmogenic cardiac disease by altering both intrinsic Ca2+-binding, as well as by dysregulating RyR2-mediated Ca2+ release via defective interaction of CaM with a distinct CaM-binding pocket that multiple RyR2 regions might contribute
Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP2 hydrolysis activity of sperm PLCĪ¶
A male infertility-linked human PLCĪ¶ (phospholipase CĪ¶) mutation introduced into mouse PLCĪ¶ completely abolishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCĪ¶ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCĪ¶, suggesting that infertility is not mediated by a dominant-negative mechanism
Hypertrophic cardiomyopathy-linked variants of cardiac myosin binding protein C3 display altered molecular properties and actin interaction
The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3ā+āV4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0āC2 protein fragments revealed that c-MYBPC3 variants alter the C0āC2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0āC2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations
Antigen unmasking enhances visualization efficacy of the oocyte activation factor, phospholipase C zeta, in mammalian sperm
Study Question
Is it possible to improve clinical visualization of phospholipase C zeta (PLCĪ¶) as a diagnostic marker of sperm oocyte activation capacity and male fertility?
Summary Answer
Poor PLCĪ¶ visualization efficacy using current protocols may be due to steric or conformational occlusion of native PLCĪ¶, hindering antibody access, and is significantly enhanced using antigen unmasking/retrieval (AUM) protocols.
What is Known Already
Mammalian oocyte activation is mediated via a series of intracellular calcium (Ca2+) oscillations induced by sperm-specific PLCĪ¶. PLCĪ¶ represents not only a potential clinical therapeutic in cases of oocyte activation deficiency but also a diagnostic marker of sperm fertility. However, there are significant concerns surrounding PLCĪ¶ antibody specificity and detection protocols.
Study Design, Size Duration
Two PLCĪ¶ polyclonal antibodies, with confirmed PLCĪ¶ specificity, were employed in mouse, porcine and human sperm. Experiments evaluated PLCĪ¶ visualization efficacy, and whether AUM improved this. Antibodies against two sperm-specific proteins [post-acrosomal WW-binding protein (PAWP) and acrosin] were used as controls.
Participants/Materials, Setting, Methods
Aldehyde- and methanol-fixed sperm were subject to immunofluorescence analysis following HCl exposure (pH = 0.1ā0.5), acid Tyrode's solution exposure (pH = 2.5) or heating in 10 mM sodium citrate solution (pH = 6.0). Fluorescence intensity of at least 300 cells was recorded for each treatment, with three independent repeats.
Main Results and the Role of Chance
Despite high specificity for native PLCĪ¶ following immunoblotting using epitope-specific polyclonal PLCĪ¶ antibodies in mouse, porcine and human sperm, immunofluorescent visualization efficacy was poor. In contrast, sperm markers PAWP and acrosin exhibited relatively impressive results. All methods of AUM on aldehyde-fixed sperm enhanced visualization efficacy for PLCĪ¶ compared to visualization efficacy before AUM (P < 0.05 for all AUM interventions), but exerted no significant change upon PAWP or acrosin immunofluorescence following AUM. All methods of AUM enhanced PLCĪ¶ visualization efficacy in mouse and human methanol-fixed sperm compared to without AUM (P < 0.05 for all AUM interventions), while no significant change was observed in methanol-fixed porcine sperm before and after. In the absence of aldehyde-induced cross-linkages, such results suggest that poor PLCĪ¶ visualization efficacy may be due to steric or conformational occlusion of native PLCĪ¶, hindering antibody access. Importantly, examination of sperm from individual donors revealed that AUM differentially affects observable PLCĪ¶ fluorescence, and the proportion of sperm exhibiting detectable PLCĪ¶ fluorescence in sperm from different males.
Limitations, Reasons for Caution
Direct correlation of fertility outcomes with the level of PLCĪ¶ in the sperm samples studied was not available. Such analyses would be required in future to determine whether the improved methodology for PLCĪ¶ visualization we propose would indeed reflect fertility status.
Wider Implications of the Findings
We propose that AUM alters conformational interactions to enhance PLCĪ¶ epitope availability and visualization efficacy, supporting prospective application of AUM to reduce misinterpretation in clinical diagnosis of PLCĪ¶-linked male infertility. Our current results suggest that it is perhaps prudent that previous studies investigating links between PLCĪ¶ and fertility parameters are re-examined in the context of AUM, and may pave the way for future work to answer significant questions such as how PLCĪ¶ appears to be kept in an inactive form in the sperm
Drug inhibition of redox factor-1 restores hypoxia-driven changes in tuberous sclerosis complex 2 deficient cells
Simple Summary: Tuberous sclerosis complex (TSC) is a genetic disease where patients are predisposed to tumors and neurological complications. Current therapies for this disease are not fully curative. We aimed to explore novel drug targets and therapies that could further benefit TSC patients. This work uncovered a novel pathway that drives disease in TSC cell models involving redox factor-1 (Ref-1). Ref-1 is a protein that turns on several key transcription factors that collectively promote tumor growth and survival through direct redox signaling. Processes regulated by Ref-1 include angiogenesis, inflammation, and metabolic transformation. Therefore, this work reveals a new drug target, where inhibitors of Ref-1 could have an additional benefit compared to current drug therapies. Abstract: Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1Ī±, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1Ī±. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1Ī± as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone
Drug inhibition of redox factor-1 restores hypoxic-driven changes in Tuberous Sclerosis Complex 2-deficient cells
Therapies with mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative
for Tuberous Sclerosis Complex (TSC) patients. Here we propose that some mTORC1-independent disease facets
of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses redox signaling activity that stimulates
the transcriptional activity of STAT3, NF-B, and HIF-1 involved in inflammation, proliferation, angiogenesis
and hypoxia, respectively. Here we demonstrate that redox signaling through Ref-1 contributes to metabolic
transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1
inhibitor, APX3330, was effective at blocking the hyperactivity of STAT3, NF-B, and HIF-1. While Ref-1
inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show
that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors.
Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant
pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis.
Therefore, this work presents Ref-1 and associated redox-regulated transcription factors, such as STAT3, NF-B
and HIF-1, as potential therapeutic targets to treat TSC, where targeting these components would likely have
additional benefits to just using mTORC1 inhibitors alone
Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours
Background
MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-Ī± in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-Ī± is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST.
Methods
We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3.
Results
MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition.
Conclusions
Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic
GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury.
Diffuse white matter injury (DWMI), a leading cause of neurodevelopmental disabilities in preterm infants, is characterized by reduced oligodendrocyte formation. NG2-expressing oligodendrocyte precursor cells (NG2 cells) are exposed to various extrinsic regulatory signals, including the neurotransmitter GABA. We investigated GABAergic signaling to cerebellar white matter NG2 cells in a mouse model of DWMI (chronic neonatal hypoxia). We found that hypoxia caused a loss of GABAA receptor-mediated synaptic input to NG2 cells, extensive proliferation of these cells and delayed oligodendrocyte maturation, leading to dysmyelination. Treatment of control mice with a GABAA receptor antagonist or deletion of the chloride-accumulating transporter NKCC1 mimicked the effects of hypoxia. Conversely, blockade of GABA catabolism or GABA uptake reduced NG2 cell numbers and increased the formation of mature oligodendrocytes both in control and hypoxic mice. Our results indicate that GABAergic signaling regulates NG2 cell differentiation and proliferation in vivo, and suggest that its perturbation is a key factor in DWMI