Molecular and structural basis of TIGIT: Nectin-4 interaction, a recently discovered pathway crucial for cancer immunotherapy.

TIGIT (T cell immunoglobulin and ITIM domain) is an inhibitory receptor expressed on T and NK cells that interact with cell surface glycoprotein belonging to the nectin and nectin-like family of cell adhesion molecules, particularly nectin-2 and nectin-like 5 (PVR). Nectin-4 has been recently identified as a novel ligand for TIGIT and the interaction among them inhibits NK cell cytotoxicity. In this study, biophysical experiments were conducted to decipher the mechanism of this novel interaction, followed by structure-guided mutagenesis studies to map the nectin-4 binding interface on TIGIT. Using surface plasmon resonance, we deduced that TIGIT recognizes the membrane distal ectodomain of nectin-4 and the interaction is weaker than the well-characterized TIGIT: nectin-2 interaction. Deciphering the molecular basis of this newly identified interaction between TIGIT and nectin-4 will provide us important insight into the manipulation of this inhibitory signaling pathway, especially targeting cancer cells overexpressing nectin-4 that evade the immune surveillance of the body.

Structural basis of molecular recognition among classical cadherins mediating cell adhesion.

Cadherins are type-I membrane glycoproteins that primarily participate in calcium-dependent cell adhesion and homotypic cell sorting in various stages of embryonic development. Besides their crucial role in cellular and physiological processes, increasing studies highlight their involvement in pathophysiological functions ranging from cancer progression and metastasis to being entry receptors for pathogens. Cadherins mediate these cellular processes through homophilic, as well as heterophilic interactions (within and outside the superfamily) by their membrane distal ectodomains. This review provides an in-depth structural perspective of molecular recognition among type-I and type-II classical cadherins. Furthermore, this review offers structural insights into different dimeric assemblies like the 'strand-swap dimer' and 'X-dimer' as well as mechanisms relating these dimer forms like 'two-step adhesion' and 'encounter complex'. Alongside providing structural details, this review connects structural studies to bond mechanics merging crystallographic and single-molecule force spectroscopic findings. Finally, the review discusses the recent discoveries on dimeric intermediates that uncover prospects of further research beyond two-step adhesion.

Heterophilic recognition between E-cadherin and N-cadherin relies on same canonical binding interface as required for E-cadherin homodimerization.

Cadherins are a family of cell surface glycoproteins that mediate Ca2+-dependent cell to cell adhesion. They organize to form large macromolecular assemblies at the junctions of cells in order to form and maintain the integrity of tissue structures, thereby playing an indispensable role in the multicellular organization. Notably, a large body of research on E- and N-cadherin, the two most widely studied members of the cadherin superfamily, suggest for homophilic associations among them to drive cell adhesion. Interestingly, latest studies also highlight for direct crosstalk among these two classical cadherins to form heterotypic connections in physiological as well as in disease environment. However, the molecular details for the heterophilic association of E-cadherin and N-cadherin has not been investigated yet, which we aimed to address in this work. Using surface plasmon resonance and flow cytometry based biophysical studies we observed heterophilic interaction between E- and N-cadherin mediated through the membrane distal ectodomains. Further, the heterodimeric interface of E-cadherin and N-cadherin was mapped using structure-guided mutational studies followed by complementary biophysical analyses to identify the important interface residues involved in the interaction. The results obtained imply significant resemblance in the interface residues of E-cadherin that are crucial for homophilic recognition of E-cadherin and heterophilic recognition of N-cadherin as well.

Cadherin-mediated host-pathogen interactions.

Cell adhesion molecules mediate cell-to-cell and cell-to-matrix adhesions and play an immense role in a myriad of physiological processes during the growth and development of a multicellular organism. Cadherins belong to a major group of membrane-bound cell surface proteins that, in coordination with nectins, drive the formation and maintenance of adherens junctions for mediating cell to cell adhesion, cellular communication and signalling. Alongside adhesive function, the involvement of cadherins in mediating host-pathogen interactions has been extensively explored in recent years. In this review, we provide an in-depth understanding of microbial pathogens and their virulence factors that exploit cadherins for their strategical invasion into the host cell. Furthermore, macromolecular interactions involving cadherins and various microbial factors such as secretory toxins and adhesins lead to the disintegration of host cell junctions followed by the entry of the pathogen or triggering downstream signalling pathways responsible for successful invasion of the pathogenic microbes are discussed. Besides providing a comprehensive insight into some of the structural complexes involving cadherins and microbial factors to offer the mechanistic details of host-pathogen interactions, the current review also highlights novel constituents of various cell signalling events such as endocytosis machinery elicited upon microbial infections.

GIpred: a computational tool for prediction of GIGANTEA proteins using machine learning algorithm.

In plants, GIGANTEA (GI) protein plays different biological functions including carbon and sucrose metabolism, cell wall deposition, transpiration and hypocotyl elongation. This suggests that GI is an important class of proteins. So far, the resource-intensive experimental methods have been mostly utilized for identification of GI proteins. Thus, we made an attempt in this study to develop a computational model for fast and accurate prediction of GI proteins. Ten different supervised learning algorithms i.e., SVM, RF, JRIP, J48, LMT, IBK, NB, PART, BAGG and LGB were employed for prediction, where the amino acid composition (AAC), FASGAI features and physico-chemical (PHYC) properties were used as numerical inputs for the learning algorithms. Higher accuracies i.e., 96.75% of AUC-ROC and 86.7% of AUC-PR were observed for SVM coupled with AAC + PHYC feature combination, while evaluated with five-fold cross validation. With leave-one-out cross validation, 97.29% of AUC-ROC and 87.89% of AUC-PR were respectively achieved. While the performance of the model was evaluated with an independent dataset of 18 GI sequences, 17 were observed as correctly predicted. We have also performed proteome-wide identification of GI proteins in wheat, followed by functional annotation using Gene Ontology terms. A prediction server "GIpred" is freely accessible at http://cabgrid.res.in:8080/gipred/ for proteome-wide recognition of GI proteins. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01130-6.

An evolutionarily conserved charged residue dictates the specificity of heterophilic interactions among nectins.

Nectins are a family of four cell surface glycoproteins belonging to the immunoglobulin superfamily that mediate cell-cell adhesion and associated signalling pathways, thereby regulating several physiological processes including morphogenesis, growth and development of multicellular organisms. Nectins interact among themselves through their extracellular domains from the adjacent cells in both homophilic and heterophilic fashions to support cell-cell adhesion. Although nectins form homodimers as demonstrated in experimental set-ups, only the specific heterophilic interactions among nectins are physiologically relevant as shown by in vivo studies. It has been hypothesised that a conserved charged residue present at the binding interface acts as the molecular switch for heterophilic nectin-nectin recognitions. In this work, we have analysed the energetics of homophilic and heterophilic interactions of nectins, followed by surface plasmon resonance-based binding studies and complementary in silico analyses. Our findings confirm that the conserved charged residues at the binding interfaces dictate the specificity of the nectin-nectin heterophilic interactions. Furthermore, these residues also play a role in conferring higher affinity to the heterophilic interactions, thereby making them physiologically more prevalent compared to homophilic interactions. Thus, this work reveals the molecular basis of heterophilic recognitions among nectins that contribute to their physiological functions.

In silico and in vitro characterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair.

Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.

Molecular Crosstalk Between Adherens Junction Proteins, E-cadherin and Nectin-4.

Cell-cell junctions formed by the association of cell adhesion molecules facilitate physiological events necessary for growth and development of multicellular organisms. Among them, cadherins and nectins organize and assemble to form adherens junction, which thereby mechanically couples interacting cells. A detailed understanding of the crosstalk involving these cell adhesion molecules is fundamental to the study of the various developmental processes. Although, cadherins and nectins can recruit each other in the adherens junction through an interplay of cytoplasmic adaptor molecules, here, we report a direct interaction between N-terminal extracellular domains of E-cadherin and nectin-4 as demonstrated by surface plasmon resonance (SPR) and Atomic Force Microscopy (AFM)-based single molecule force spectroscopy (SMFS). Kinetic studies using SPR demonstrate the binding between the ectodomains of E-cadherin and nectin-4 with a KD of 3.7 ± 0.7 µM and KD of 5.4 ± 0.2 µM (reciprocal experiment). AFM-based SMFS experiments also support interaction between the ectodomains of E-cadherin and nectin-4 with the koff value of 31.48 ± 1.53 s-1 and the lifetime of the complex of 0.036 ± 0.0026 s. We thus propose a cell adhesion mechanism mediated by E-cadherin and nectin-4, which can have functional significance in early embryogenesis as evident from the expression pattern of both the proteins during early development.

High-resolution crystal structure of LpqH, an immunomodulatory surface lipoprotein of Mycobacterium tuberculosis reveals a distinct fold and a conserved cleft on its surface.

Tuberculosis, caused by Mycobacterium tuberculosis, is predominantly a disease of the lungs acquired by inhaling mycobacteria from infected individuals via airborne droplets. In order to facilitate their entry into the alveolar macrophages, mycobacteria have a collection of pathogen-associated molecular patterns (PAMPs) on their surface that are known to detect certain pattern recognition receptors present on the surface of host cells. A major group of these PAMPs includes mycobacterial lipoproteins, of which, the 19 kDa surface antigen LpqH, has been reported to play a critical role in both host-pathogen interactions as well as pleiotropic immune regulation. Despite its crucial involvement in tuberculosis, the detailed structure-function relationship of this protein remains to be explored. Here, we report the high-resolution crystal structure of the non-acylated LpqH (LpqH48-159) at a resolution of 1.26 Å, which adopts a unique fold. Flow cytometry-based experiments show that the protein can bind and induce apoptosis in PMA-activated human monocytic cell line THP-1, indicative of the preservation of functionality of the protein. Furthermore, analysis of conservation of LpqH sequences from Mycobacterium species reveals a patch of conserved residues on the surface which may play a role in its binding partner recognition and hence in host-pathogen interaction.

Molecular modeling and co-expression analysis of human stem cell factor as fusion partner to granulocyte colony stimulating factor for improving their bioactivity.

Human granulocyte colony stimulating factor (hG-CSF) is an expensive hematopoietic growth factor that is clinically used in human for the treatment of neutropenia in diseases such as AIDS, aplastic anemia, myelodysplastic syndrome and congenital or chemotherapy-induced neutropenia. Here, through a computational biology approach, we show that human stem cell factor (hSCF) could be a better fusion partner than human thyroid peroxidase (hTPO), human erythropoietin (hEPO) and human interleukin-3 (hIL3) for co-expression with hG-CSF. Molecular modeling of hG-CSF-hSCF fusion protein with hG-CSF and hSCF receptors showed that binding of fusion protein with human granulocyte colony stimulating factor receptor (hG-CSFR) did not inhibit its binding to human stem cell factor receptor (hSCFR) and vice versa. To validate the results, coding sequences of hG-CSF and hSCF were cloned and co-expressed as fusion protein and their bioactivity was evaluated on hG-CSF responsive 3T3 cell line. The fused expression vector expressed recombinant hG-CSF-hSCF upon IPTG-induction, as revealed by real-time polymerase chain reaction (RT-PCR), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Bioactivity analysis confirmed that rhG-CSF-hSCF protein had higher bioactivity than hG-CSF. Thus, hSCF could be a good fusion partner for hG-CSF and its co-expression as hG-CSF-hSCF may offer an alternative to individual use of two hematopoietic factors in clinics. Future studies should determine the purification strategies, folding status and mechanism of action of the recombinant proteins. Communicated by Ramaswamy H. Sarma.

In vitro α-amylase and α-glucosidase Inhibition, Antioxidant, Anti- Inflammatory Activity and GC-MS Profiling of Avicennia alba Blume.

BACKGROUND: Avicennia alba Blume, is a well-known mangrove plant used in traditional medicinal practices for several human ailments. OBJECTIVE: The study aimed at the evaluation of antidiabetic, antioxidant, anti-inflammatory and cytotoxic activities of A. alba ethanolic leaf (AAL) and bark (AAB) extract along with phytochemical investigation. METHODS: In vitro antidiabetic study was done by α-amylase, α-glucosidase enzyme inhibition assay; antioxidant study by DPPH, ABTS, superoxide, and metal chelating assays, antiinflammatory study by protein denaturation assay. The cytotoxicity study was done on TC1 murine cell line. Further, GC-MS analysis was carried out for AAL extracts. RESULTS: AAL exhibited better antidiabetic activities with IC50 values of 1.18 and 0.87 mg/ml against α-amylase and α-glucosidase enzymes respectively. The AAL exhibited better ABTS, superoxide scavenging and metal chelating potential with IC50 values of 0.095, 0.127 and 0.444 mg/ml. However, AAB showed higher DPPH scavenging potential with IC50 value of 0.163 mg/ml. The AAL also exhibited higher protein denaturation potential with IC50 value of 0.370 mg/ml. The bark extract exhibited better cytotoxic activity as compared to leaf extracts on the TC1 murine cell line. The phytochemical study revealed higher total phenol (25.64 mg GAE/g), flavonoid (205.09 mg QE/g), and tannin content (251.17 mg GAE/g) in AAL. The GC-MS analysis revealed the presence of several compounds in AAL extract. CONCLUSION: The result of the present study highlights the antidiabetic, antioxidant and cytotoxic activities of mangrove plant Avicennia alba.