Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca release via the inositol 1,4,5-trisphosphate (InsP) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca oscillations within mammalian eggs. Moreover, studies describing PLCζ "knockout" mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca oscillations and egg activation in mammals, while also discussing the nature of a potential "alternative" sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.Qatar University student grant QUST-1-CMED-2020-3. Healthcare Research Fellowship Award (HF-14-16) made by Health and Care Research Wales (HCRW). National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST)

Calmodulin Interacts and Regulates Enzyme Activity of the Mammalian Sperm Phospholipase C

Sperm-specific Phospholipase C zeta (PLCζ) 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. PLCζ, 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 PLCζ 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 PLCζ. Bioinformatics analysis revealed putative CaM-binding sites on PLCζ sequence. In the present study, we have used co-immunoprecipitation analysis and we show that in the presence of Ca2+, human PLCζ directly interacts with CaM. Isothermal titration calorimetry (ITC) experiments were performed to map the interaction. Three different peptides corresponding to disparate sequences within human PLCζ were used and it was shown that PLCζ 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 PLCζ in the presence of Ca2+, only through one of its lobe domains. In vitro PIP2 hydrolysis assays revealed that CaM alters PLCζ PIP2 hydrolytic activity at high Ca2+ concentrations and, as suggested by liposome binding assays, this appears to be due to CaM binding to PLCζ 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 μM, 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

Arrhythmogenic calmodulin E105A mutation alters cardiac RyR2 regulation leading to cardiac dysfunction in zebrafish

Calmodulin (CaM) is a universal calcium (Ca2+)‐binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates excitation–contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life‐threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS‐associated missense CaM mutation (E105A) was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+‐binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM–RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–RyR2 interactions

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 (V3 + V4), 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 C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 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 C0–C2 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

Study of structural changes in protein by differential scanning calorimetry: structure-based thermodynamic stability of the BRCT domain of BRCA1, BARD1 and 53BP1

Tandem BRCT repeats are phosphopeptide-binding modules found in several proteins that participate in transcriptional activation networks, DNA-damage checkpoint control and genomic stability. The BRCA1-associated RING domain protein 1 (BARD1) is a 777 amino acid-long protein that contains a RING finger, three ankyrin repeats and the BRCT domain. It is the major binding partner of the breast and ovarian tumor suppressor BRCA1, functioning by forming a heterodimer with BRCA1. 53BP1 is another protein with a BRCT domain that plays an important role in the cellular response to DNA damage. 53BP1 interacts with the DNA-binding core domain of the tumor suppressor p53 and enhances p53-mediated transcriptional activation. The interaction between p53 and 53BP1 is mediated by the C-terminal BRCT domain. It has been shown that thermodynamic destabilization of the BRCT domain of BRCA1 is induced by missense mutations linked to hereditary breast-ovarian cancer. To gain insight into the differential thermodynamic stability of the BRCT domains of BRCA1, BARD1 and 53BP1, a series of biophysical techniques were employed to analyze their thermodynamic stability by chemical and thermal unfolding. To this end recombinant 6xHis fusion tagged BARD1 and 53BP1- BRCT proteins were overexpressed in E. coli. To enable the characterization of these proteins via calorimetric and spectroscopic studies, the 6xHis fusion tag was enzymatically removed and the native proteins were purified to homogeneity. These proteins have been subjected to circular dichroism, differential scanning calorimetry and fluorescence spectroscopy. Finally, to investigate whether the BARD1- BRCT domain interacts with the BRIP1 phosphopeptide, a series of isothermal titration experiments was performed under solution conditions that maximize the stability of the domain. Interestingly, in 50 mM Borate pH 9.0, 300 mM NaCl and at 10°C, it was possible to record an interaction with the BRIP1 phosphopeptide. This is the first report of an interaction of the BARD1- BRCT domain with a pSer-X-X-Phe motif phosphopeptide, a result which underlines the major role of thermodynamic stability in protein-protein interactions and explains the different binding preferences of these BRCT domains.Οι επαναλήψεις BRCT είναι δομικά πρωτεϊνικά στοιχεία με ικανότητα δέσμευσης φωσφοπεπτιδίων που εμφανίζονται συχνά σε πρωτεΐνες που συμμετέχουν στα δίκτυα ενεργοποίησης της μεταγραφής, σε κρίσιμα σημεία ελέγχου βλαβών του DNA και στη διατήρηση της σταθερότητας του γονιδιόματος. Η BARD1 είναι μια πρωτεΐνη 777 αμινοξικών καταλοίπων που περιέχει μια RING δομική περιοχή, τρείς επαναλήψεις αγκυρίνης και μια περιοχή BRCT. Είναι η πιο σημαντική από τις πρωτεΐνες που αλληλεπιδρούν με το ογκοκατασταλτικό μόριο BRCA1, μόριο με το οποίο σχηματίζει ένα ετεροδιμερές με μεγάλη βιολογική δραστικότητα. Η 53BP1 είναι μια ακόμα πρωτεΐνη που διαθέτει BRCT δομική περιοχή και που διαδραματίζει έναν σημαντικό ρόλο στις κυτταρικές αποκρίσεις που ακολουθούν τον εντοπισμό βλαβών στο DNA. Η 53BP1 αλληλεπιδρά με τη DNA-δεσμευτική περιοχή της ογκοκατασταλτικής πρωτεΐνης p53 και ενισχύει την p53-εξαρτώμενη μεταγραφική ενεργοποίηση. Η αλληλεπίδραση μεταξύ της p53 και της 53BP1 πραγματοποιείται μέσω της BRCT περιοχής της τελευταίας. Έχει διαπιστωθεί ότι η θερμοδυναμική αποσταθεροποίηση της περιοχής BRCTBRCA1, σαν αποτέλεσμα της παρουσίας παρανοηματικών μεταλλαγών στο μόριο, συνδέεται με τον κληρονομικό καρκίνο μαστού-ωοθηκών. Με στόχο την αποτίμηση της θερμοδυναμικής σταθερότητας των περιοχών BRCT των πρωτεϊνών BARD1 και 53BP1, πραγματοποιήθηκε μια σειρά βιοφυσικών πειραμάτων για την ανάλυση της θερμικά και χημικά επαγόμενης αποδιάταξης των μορίων αυτών. Αρχικά οι επισημασμένες με ετικέτα ιστιδινών (6xHis) πρωτεΐνες BARD1 και 53BP1-BRCT υπερεκφράστηκαν σε καλλιέργειες βακτήριων Ε. coli. Για τη πραγματοποίηση θερμιδομετρικών και φασματοσκοπικών μελετών κρίθηκε απαραίτητη η αποκοπή της ετικέτας ιστιδινών, η οποία και πραγματοποιήθηκε ενζυμικά και στη συνέχεια ακολούθησε απομόνωση και καθαρισμός των πρωτεϊνικών μορίων. Οι πρωτεΐνες αυτές μελετήθηκαν με κυκλικό διχροϊσμό, αδιαβατική μικροθερμιδομετρία διαφορικής σάρωσης και φασματοσκοπία φθορισμού. Τέλος, προκειμένου να ερευνηθεί εάν η περιοχή BARD1-BRCT αλληλεπιδρά με το φωσφοπεπτίδιο BRIP1, μια σειρά πειραμάτων θερμιδομετρίας ισόθερμης τιτλοδότησης πραγματοποιήθηκε σε συνθήκες που μεγιστοποιούν τη σταθερότητα της δομικής αυτής περιοχής. Κατά τρόπο ενδιαφέροντα, σε ρυθμιστικό διάλυμα 50 mM βορικών pH 9.0, 300 mM ΝaCl και σε θερμοκρασία 10°C, ήταν δυνατό να καταγραφεί αλληλεπίδραση της πρωτεΐνης BARD1-BRCT με το φωσφοπεπτίδιο BRIP1. Αυτή είναι η πρώτη αναφορά στη διεθνή βιβλιογραφία μιας αλληλεπίδρασης της BARD1-BRCT περιοχής με φωσφοπεπτίδιο δομικού μοτίβου pSerX-X-Phe, ένα αποτέλεσμα που υπογραμμίζει το σημαντικό ρόλο της θερμοδυναμικής σταθερότητας στις πρωτεϊνικές-πρωτεϊνικές αλληλεπιδράσεις και εξηγεί τις διαφορές που παρουσιάζουν οι περιοχές αυτές όσον αφορά τη δέσμευση φωσφοπεπτιδίων

ApoM binds endotoxin contributing to neutralization and clearance by High Density Lipoprotein

Background: HDL possesses anti-inflammatory properties, however, the exact mechanism is not fully understood. Endotoxin is a potent inducers of TLR4 signaling, leading to inflammatory mediators’ release. It has been estimated that TLR4 recognizes about 5% of circulating lipopolysaccharide whereas 95% is cleared by plasma lipoproteins, mainly HDL. ApoM is required for HDL biogenesis and 95% of plasma ApoM is found associated with HDL, both are significantly reduced during sepsis. Aim: The aim of this study is to investigate whether ApoM binds endotoxin and contributes to anti-inflammatory activity of HDL. Methods: Isothermal Titration Calorimetry (ITC) was used to determine the binding of ultrapure E. coli LPS to the recombinant ApoM protein. Purified human HDL and recombinant ApoM was used to investigate LPS neutralization using human and murine macrophages and computational simulation was performed. Result: ApoM shows high affinity for E. coli LPS, forming 1:1 complexes with Kd values below 1 μΜ, as revealed by ITC. The binding process is strongly exothermic and enthalpy-driven (ΔrH = −36.5 kJ/mol), implying the formation of an extensive network of interactions between ApoM and LPS in the bound state. Computational simulation also predicted high-affinity binding between ApoM and E. coli LPS and the best scoring models showed E. coli LPS docking near the calyx of ApoM without blocking the pocket. The biological significance of this interaction was further demonstrated in macrophages where purified HDL neutralized an E. coli LPS effect and significantly reduced TNFα release from human THP-1 cells. Conclusion: ApoM binds LPS to facilitate endotoxin neutralization and clearance by HDL

ApoM binds endotoxin contributing to neutralization and clearance by High Density Lipoprotein.

HDL possesses anti-inflammatory properties, however, the exact mechanism is not fully understood. Endotoxin is a potent inducers of TLR4 signaling, leading to inflammatory mediators' release. It has been estimated that TLR4 recognizes about 5% of circulating lipopolysaccharide whereas 95% is cleared by plasma lipoproteins, mainly HDL. ApoM is required for HDL biogenesis and 95% of plasma ApoM is found associated with HDL, both are significantly reduced during sepsis. The aim of this study is to investigate whether ApoM binds endotoxin and contributes to anti-inflammatory activity of HDL. Isothermal Titration Calorimetry (ITC) was used to determine the binding of ultrapure LPS to the recombinant ApoM protein. Purified human HDL and recombinant ApoM was used to investigate LPS neutralization using human and murine macrophages and computational simulation was performed. ApoM shows high affinity for LPS, forming 1:1 complexes with Kd values below 1 μΜ, as revealed by ITC. The binding process is strongly exothermic and enthalpy-driven (ΔH = -36.5 kJ/mol), implying the formation of an extensive network of interactions between ApoM and LPS in the bound state. Computational simulation also predicted high-affinity binding between ApoM and LPS and the best scoring models showed LPS docking near the calyx of ApoM without blocking the pocket. The biological significance of this interaction was further demonstrated in macrophages where purified HDL neutralized an LPS effect and significantly reduced TNFα release from human THP-1 cells. ApoM binds LPS to facilitate endotoxin neutralization and clearance by HDL

From Nucleobases to Nucleolipids: An ITC Approach on the Thermodynamics of Their Interactions in Aqueous Solutions

Hybrid constructions based on nucleosides and lipophilic components, known as nucleolipids, have become an extremely interesting class of molecules, especially for their potential biomedical applications. In this matter, it seemed important to define the nature and estimate the strength of their interaction with polynucleotides by different ways. We report in this work a systematic investigation through isothermal titration calorimetry of the thermodynamics of the association and dissociation of adenine and thymine derivatives, not previously performed...

Orientation of biotin-binding sites in streptavidin adsorbed onto the surface of polythiophene films

The orientation of biotin-binding sites of streptavidin adsorbed to thin films of three polythiophenes (PTs), namely, regioregular poly­(3-hexylthiophene) (RP3HT), regiorandom poly­(3-butylthiophene) (P3BT), and poly­(3,3‴-didodecylquaterthiophene) (PQT12), has been investigated. Polymer films were examined prior to and after protein adsorption with atomic force microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA) applied to ToF-SIMS data revealed subtle changes in surface chemistry of polymer films and orientation of adsorbed streptavidin. PCA resolved the surface alignment of alkyl side chains and differentiated the ToF-SIMS data for PQT12, RP3HT, and P3BT, verifying an amorphous morphology for P3BT and a semicrystalline one for PQT12 and RP3HT. After the characterization of the polymeric films, streptavidin adsorption from solutions with different protein concentrations (up to 300 μg/mL) has been conducted. The PCA results distinguished between amino acids characteristic for external regions of streptavidin molecules adsorbed to different PTs suggest that streptavidin adsorbed to PQT12 exposes molecular regions rich in tryptophan and tyrosine, which are components of the biotin-binding sites. The latter results were confirmed using biotin-labeled horse radish peroxidase to estimate the exposed binding sites of streptavidin adsorbed onto the different PT films. The analysis of streptavidin structure suggests that interaction between polythiophene film and dipole moment of streptavidin subunit is responsible for orientation of biotin-binding sites