Characterizing the Conformational Dynamics of Human SUMO2: Insights into its Interaction with Metal Ions and SIMs.

SUMO (Small Ubiquitin-like Modifiers) proteins are involved in a crucial post-translational modification commonly termed as SUMOylation. In this work, we have investigated the native-state conformational flexibility of human SUMO2 and its interaction with Cu2+ and Zn2+ ions using 15N-1H based 2D NMR spectroscopy. After SUMO1, SUMO2 is the most studied SUMO isoform in humans which shares 45 % and ~80 % similarity with SUMO1 in terms of sequence and structure, respectively. In this manuscript, we demonstrate that compared to SUMO1, several amino acids around the α1-helix region of SUMO2 access energetically similar near-native conformations. These conformations could play a crucial role in SUMO2's non-covalent interactions with SUMO interaction motifs (SIMs) on other proteins. The C-terminal of SUMO2 was found to bind strongly with Cu2+ ions resulting in a trimeric structure as observed by gel electrophoresis. This interaction seems to interfere in its non-covalent interaction with a V/I-x-V/I-V/I based SIM in Daxx protein.

How 1,n-Bis(3-alkylimidazolium-1-yl) Alkane Interacts with the Phospholipid Membrane and Impacts the Toxicity of Dicationic Ionic Liquids.

Ionic liquids based on doubly charged cations, often termed dicationic ionic liquids (DILs), offer robust physicochemical properties and low toxicity than conventional monocationic ionic liquids. In this design-based study, we used solid-state NMR spectroscopy to provide the interaction mechanism of two DILs, 1,n-bis(3-alkylimidazolium-1-yl) alkane dibromide ([C2n(C7-nIM)2]2+·2Br-, n = 1, 6), with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipid membranes, to explain the low toxicity of DILs toward HeLa, Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae cell lines. Dications with a short linker and long terminal chains cause substantial perturbation to the bilayer structure, making them more membrane permeabilizing, as shown by fluorescence-based dye leakage assays. The structural perturbation is even higher than [C12(MIM)]+ monocations, which carry a single 12-carbon long chain and exhibit a much higher membrane affinity, permeability, and cytotoxicity. These structural details are a crucial contribution to the design strategies aimed at harnessing the biological activity of ionic liquids.

A Comparative Analysis of the Altered Levels of Human Seminal Plasma Constituents as Contributing Factors in Different Types of Male Infertility.

(1) Background: The relationships between the biochemical and immunological components in seminal plasma and their physiological effects on male reproductive system have been underreported. In this study, we evaluated the potential of several seminal plasma biochemical and immunological markers in the pathophysiological developments of the infertile male patients. The study was designed to identify and assess different markers that may be associated with semen functions in different types of male infertility. (2) Methods: A total of 50 infertile male patients who underwent checkup for fertility assessment and 50 fertile controls were included in this study. The complete medical history of each recruited participant was reviewed. The infertile sub-groups (non-obstructive azoospermia (NOA), asthenozoospermia (AS), normozoospermic infertile (NI), and oligozoospermia (OZ)) were characterized based on sperm motility and concentration, while NI patients were included after a thorough check up of their female partners as well. We investigated each sample for 21 different analytes, enzymes, trace elements, and immunological markers to find crucial markers posing as contributing factors to a specific type of male infertility. (3) Results: The levels of 15 out of 21 markers, assayed from the seminal plasma of infertile males, were significantly altered in comparison to fertile controls (p < 0.05). For the first time, microprotein levels were also analyzed. The presence of monocytes, lymphocytes, and granulocytes was limited to semen from NOA patients, while a significant increase in the level of platelets was observed in AS. Hierarchical clustering and ROC-AUC analysis identified the three most significant markers (zinc, LDH, and TG) for the healthy control group and asthenozoospermic group (AUC, of 0.92 and 0.81, respectively). (4) Conclusions: The altered levels of biochemical and immunological markers in seminal plasma might be associated with the different male infertility profiles and could be required for the sperm metabolism and maintenance. However, a larger sample size and follow up analysis is required for establishing the hypothesized panel of markers as biomarkers at clinical stage.

The enigmatic sperm proteins in mammalian fertilization: an overview†.

Mammalian fertilization involves a physical interaction between a sperm and an egg followed by molecular interactions amongst their various cell surface molecules. These interactions are initially mediated on the egg's outermost matrix, zona pellucida (ZP), and then its plasma membrane. To better understand this process, it is pertinent to find the corresponding molecules on sperm that interact with ZP or the egg's plasma membrane. Although currently, we have some knowledge about the binding partners for egg's plasma membrane on sperm, yet the ones involved in an interaction with ZP have remained remarkably elusive. This review provides comprehensive knowledge about the various sperm proteins participating in mammalian fertilization and discusses the possible reasons for not being able to identify the strong sperm surface candidate (s) for ZP adhesion. It also hypothesizes the existence of a multi-protein complex(s), members of which participate in oviduct transport, cumulus penetration, zona adhesion, and adhesion/fusion with the egg's plasma membrane; with some protein(s) having multiple roles during this process. Identification of these proteins is crucial as it improves our understanding of the process and allows us to successfully treat infertility, develop contraceptives, and improve artificial reproductive technologies.

Effect of the Alkyl Chain Length of Amphiphilic Ionic Liquids on the Structure and Dynamics of Model Lipid Membranes.

We compare the biophysical and structural aspects of the interaction of amphiphilic ionic liquids containing 1-alkyl-3-methylimidazolium cation ([CnMIM]+, n = 8, 12, or 16) with membranes composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG). Liposome affinity and permeabilization were determined using ζ-potential and fluorescence studies, correlated with the cytoxicity of [CnMIM]+Br- toward HeLa cell lines. Membrane affinity is strongest in the case of [C16MIM]+Br- followed by [C12MIM]+Br- and [C8MIM]+Br- for both membranes, and trends remained the same in the case of membrane permeability and cytotoxicity. Solid-state NMR spectroscopy was used to localize [CnMIM]+ inside the lipid bilayers and to study their impact on the head group and acyl chain structures and dynamics of the lipid molecules. The charged ring moiety of the [CnMIM]+ is localized in the lipid-water interface of the membranes irrespective of the chain length and membrane surface charge. While [C8MIM]+ binds the membrane most weakly, it induces the largest disorder in the lipid chain region. A lack of fast flip-flop motions of the amphiphiles in the case of long chain [C16MIM]+ is suggested to render the membrane unstable, which increases its permeability. Between the lipid molecules, the POPC membrane incurs larger disorder in lipid chain packing upon insertion of [CnMIM]+ molecules. The study provides structural details of the impact of increasing chain lengths in [CnMIM]+ on the structural properties of lipid bilayers.

Understanding seminal plasma in male infertility: emerging markers and their implications.

Infertility affects a significant proportion of the reproductive-aged population, with male-associated factors contributing to over half of the cases. However, current diagnostic tools have limitations, leading to an underestimation of the true prevalence of male infertility. While traditional semen parameters provide some insights, they fail to determine the true fertility potential in a substantial number of instances. Therefore, it is crucial to investigate additional molecular targets responsible for male infertility to improve understanding and identification of such cases. Seminal plasma, the main carrier of molecules derived from male reproductive glands, plays a crucial role in reproduction. Amongst its multifarious functions, it regulates processes such as sperm capacitation, sperm protection and maturation, and even interaction with the egg's zona pellucida. Seminal plasma offers a non-invasive sample for urogenital diagnostics and has shown promise in identifying biomarkers associated with male reproductive disorders. This review aims to provide an updated and comprehensive overview of seminal plasma in the diagnosis of male infertility, exploring its composition, function, methods used for analysis, and the application of emerging markers. Apart from the application, the potential challenges of seminal plasma analysis such as standardisation, marker interpretation and confounding factors have also been addressed. Moreover, we have also explored future avenues for enhancing its utility and its role in improving diagnostic strategies. Through comprehensive exploration of seminal plasma's diagnostic potential, the present analysis seeks to advance the understanding of male infertility and its effective management.

Identification and in silico Characterization of Deleterious Single Nucleotide Variations in Human ZP2 Gene.

ZP2, an important component of the zona matrix, surrounds mammalian oocytes and facilitates fertilization. Recently, some studies have documented the association of mutations in genes encoding the zona matrix with the infertile status of human females. Single nucleotide polymorphisms are the most common type of genetic variations observed in a population and as per the dbSNP database, around 5,152 SNPs are reported to exist in the human ZP2 (hZP2) gene. Although a wide range of computational tools are publicly available, yet no computational studies have been done to date to identify and analyze structural and functional effects of deleterious SNPs on hZP2. In this study, we conducted a comprehensive in silico analysis of all the SNPs found in hZP2. Six different computational tools including SIFT and PolyPhen-2 predicted 18 common nsSNPs as deleterious of which 12 were predicted to most likely affect the structure/functional properties. These were either present in the N-term region crucial for sperm-zona interaction or in the zona domain. 31 additional SNPs in both coding and non-coding regions were also identified. Interestingly, some of these SNPs have been found to be present in infertile females in some recent studies.

Cytotoxicity and Membrane Permeability of Double-Chained 1,3-Dialkylimidazolium Cations in Ionic Liquids.

We have evaluated ionic liquids based on double-chained 1-alkyl-3-octylimidazolium cations ([CnC8IM]+, n = 2, 4, 6, 8, 10, 12) for their cytotoxicity toward various cell lines. The toxicity of ionic liquids was correlated to their ability to partition into and permeabilize phosphocholine (POPC)- or phosphoglycerol (POPG)-based large unilamellar vesicles. Membrane partitioning of ionic liquids was assessed using the ζ-potential measurements, and membrane permeability was determined using fluorescence-based dye leakage assays. Both cytotoxicity and membrane permeability of these ILs were found to increase in a sigmoidal fashion with increasing chain length on the N1 atom (n in [CnC8IM]+) cations. These results were compared with those for ionic liquids based on single-chained 1-alkyl-3-methylimidazolium cations ([Cn+8C1IM]+), carrying a similar number of carbon atoms but as a single alkyl chain. Our studies show that ionic liquids containing double-chained cations are relatively less cytotoxic and membrane-permeabilizing than the cations bearing a single long alkyl chain.

Role of cationic head-group in cytotoxicity of ionic liquids: Probing changes in bilayer architecture using solid-state NMR spectroscopy.

The effect of cationic head-group of ionic liquid on the structure and dynamics of phospholipid bilayer was studied to provide insights into the mechanism of ionic liquid-membrane interaction. The effect was observed using six ionic liquids containing benzimidazolium, imidazolium, pyrrolidinium, piperidinium, ammonium, and morpholinium based amphiphilic cations carrying a dodecyl alkyl chain. Unilamellar and multilamellar vesicles composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used. Permeability of POPC bilayer was found to have a strong dependence on ionic liquid head-group structure. To probe the structural details of interaction, 31P and 2H based solid-state NMR measurements were performed. The cations differed in terms of their effect on the orientation and disorder in the phosphocholine moiety in lipid head-group as revealed by chemical shift anisotropy of 31P. Cations carrying an unshielded charge like benzimidazolium, imidazolium, and ammonium result in strong reorientation of phosphocholine moiety in lipid head-group. Large sized cations like benzimidazolium and piperidinium result in enhanced lipid chain dynamics as revealed by order parameter calculations of deuterated lipid chains. Relatively polar head-group of morpholinium cation neither impacts the phospholipid head-group nor chain packing. Our results suggest that there exists a direct correlation between ionic liquid head-group induced structural changes in bilayer and their ability to permeabilize/disrupt the membrane and be cytotoxic.

Characterization of Cu2+ and Zn2+ binding sites in SUMO1 and its impact on protein stability.

Metal ions like Cu2+ and Zn2+ have been shown to impact protein misfolding pathways in neurodegenerative proteinopathies like Alzheimer's and Parkinson's. Also, due to their strong interaction with Ubiquitin, they interfere in degradation of misfolded proteins by impairing the ubiquitin-proteasome system (UPS). In this work, we have studied the interaction of these metal ions with a small Ubiquitin like post-translation modifier SUMO1, which is known to work co-operatively with Ubiquitin to regulate UPS system. Between Cu2+ and Zn2+, the former binds more strongly with SUMO1 as determined using fluorescence spectroscopy. SUMO1 aggregates, forming trimer and higher oligomers in presence of Cu2+ ions which were characterized using gel electrophoresis, Bradford assay, and transmission electron microscopy. Chemical shift analysis using 15N/1H based NMR spectroscopy revealed that SUMO1 retains its structural fold in its trimeric state. Cu2+ induced paramagnetic quenching and Zn2+ induced chemical shift perturbation of 15N-1H cross-peaks were used to identify their respective binding sites in SUMO1. Binding sites so obtained were further validated with molecular dynamics studies. Our findings provide structural insights into the SUMO1-Cu2+/Zn2+ interaction, and its impact on aggregation of SUMO1 which might affect its ability to modify functions of target proteins.

Molecular interaction between human SUMO-I and histone like DNA binding protein of Helicobacter pylori (Hup) investigated by NMR and other biophysical tools.

The proteins secreted by bacteria contribute to immune mediated gastric inflammation and epithelial damage; thus aid bacterial invasion in host tissue, and may also interact with host proteins, conspirating a mechanism against host-immune system. The Histone-like DNA binding protein is one of the most abundant nucleoid-associated proteins in Helicobacter pylori (H. pylori). The protein -referred here as Hup- is also secreted in vitro by H. pylori, thus it may have its role in disease pathogenesis. This is possible only if Hup interact with some human proteins including Small-Ubiquitin-like-Modifier (SUMO) proteins. Studies have established that SUMO-proteins participate in various innate-immune pathways and thus promote an efficient immune response to combat pathogenic infections. Sequence analysis revealed the presence of two SUMO interacting motifs (SIMs) and several positively charged lysine residues on the protein surface of Hup. Additionally, SUMO-proteins epitomize negatively charged surface which confers them the ability to bind to DNA/RNA binding proteins. Based on the presence of SIMs as well as charge complementarity between the proteins, it is legitimate to consider that Hup protein would bind to SUMO-proteins. The present study has been undertaken to establish this interaction for the first time using NMR in combination with ITC and other biophysical techniques.

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis.

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.