Structural insights into pharmacophore-assisted in silico identification of protein–protein interaction inhibitors for inhibition of human toll-like receptor 4 – myeloid differentiation factor-2 (hTLR4−MD-2) complex

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS?TLR4?MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein–protein interaction (PPI) in TLR4?MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4?MD-2) and dimerization (MD-2?TLR4*) protein–protein interaction interfaces in TLR4?MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4?MD-2 protein–protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in μM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4?MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.     

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional ‘single target–single drug’ approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3’-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC₅₀ and EC₅₀ values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.     

Why did only one genus of insects,Halobates, take to the high seas?

Oceans cover more than 70% of the Earth’s surface and house a dizzying array of organisms. Mammals, birds, and all manner of fish can be commonly sighted at sea, but insects, the world’s most common animals, seem to be completely absent. Appearances can deceive, however, as 5 species of the ocean skater Halobates live exclusively at the ocean surface. Discovered 200 years ago, these peppercorn-sized insects remain rather mysterious. How do they cope with life at the ocean surface, and why are they the only genus of insects to have taken to the high seas?

Oceans cover over 70% of the earth’s surface and house a dizzying array of organisms, including five species of the peppercorn-sized ocean-skater Halobates, which live exclusively at the ocean surface. How do they cope with life at the ocean surface and why are they the only genus of insects able to conquer the high seas?     

Fishing cat Prionailurus viverrinus distribution and habitat suitability in Nepal

The fishing cat Prionailurus viverrinus is a wetland specialist species endemic to South and Southeast Asia. Nepal represents the northern limit of its biogeographic range, but comprehensive information on fishing cat distribution in Nepal is lacking. To assess their distribution, we compiled fishing cat occurrence records (n = 154) from Nepal, available in published literature and unpublished data (2009–2020). Bioclimatic and environmental variables associated with their occurrence were used to predict the fishing cat habitat suitability using MaxEnt modeling. Fishing cat habitat suitability was associated with elevation (152–302 m), precipitation of the warmest quarter, i.e., April–June (668–1014 mm), precipitation of the driest month (4–7 mm), and land cover (forest/grassland and wetland). The model predicted an area of 4.4% (6679 km²) of Nepal as potential habitat for the fishing cat. About two‐thirds of the predicted potentially suitable habitat lies outside protected areas; however, a large part of the highly suitable habitat (67%) falls within protected areas. The predicted habitat suitability map serves as a reference for future investigation into fishing cat distribution as well as formulating and implementing effective conservation programs in Nepal. Fishing cat conservation initiatives should include habitats inside and outside the protected areas to ensure long‐term survival. We recommend conservation of wetland sites, surveys of fishing cats in the identified potential habitats, and studying their genetic connectivity and population status.     

Genetics of osteopontin in patients with chronic kidney disease: The German Chronic Kidney Disease study

Osteopontin (OPN), encoded by SPP1, is a phosphorylated glycoprotein predominantly synthesized in kidney tissue. Increased OPN mRNA and protein expression correlates with proteinuria, reduced creatinine clearance, and kidney fibrosis in animal models of kidney disease. But its genetic underpinnings are incompletely understood. We therefore conducted a genome-wide association study (GWAS) of OPN in a European chronic kidney disease (CKD) population. Using data from participants of the German Chronic Kidney Disease (GCKD) study (N = 4,897), a GWAS (minor allele frequency [MAF]≥1%) and aggregated variant testing (AVT, MAF<1%) of ELISA-quantified serum OPN, adjusted for age, sex, estimated glomerular filtration rate (eGFR), and urinary albumin-to-creatinine ratio (UACR) was conducted. In the project, GCKD participants had a mean age of 60 years (SD 12), median eGFR of 46 mL/min/1.73m² (p25: 37, p75: 57) and median UACR of 50 mg/g (p25: 9, p75: 383). GWAS revealed 3 loci (p<5.0E-08), two of which replicated in the population-based Young Finns Study (YFS) cohort (p<1.67E-03): rs10011284, upstream of SPP1 encoding the OPN protein and related to OPN production, and rs4253311, mapping into KLKB1 encoding prekallikrein (PK), which is processed to kallikrein (KAL) implicated through the kinin-kallikrein system (KKS) in blood pressure control, inflammation, blood coagulation, cancer, and cardiovascular disease. The SPP1 gene was also identified by AVT (p = 2.5E-8), comprising 7 splice-site and missense variants. Among others, downstream analyses revealed colocalization of the OPN association signal at SPP1 with expression in pancreas tissue, and at KLKB1 with various plasma proteins in trans, and with phenotypes (bone disorder, deep venous thrombosis) in human tissue. In summary, this GWAS of OPN levels revealed two replicated associations. The KLKB1 locus connects the function of OPN with PK, suggestive of possible further post-translation processing of OPN. Further studies are needed to elucidate the complex role of OPN within human (patho)physiology.     

Interleukin-5-mediated allergic airway inflammation inhibits the human surfactant protein C promoter in transgenic mice

Allergen challenge in the lung of humans and animals is associated with surfactant dysfunction, but the mechanism of this effect has not been established. By using a murine model of asthma we now report the effect of allergen-induced airway inflammation on the expression of transgenes regulated by the human surfactant protein (hSP)-C promoter. The hSP-C 3.7-kilobase pair promoter was used to direct the expression of eotaxin, an eosinophil-selective chemokine, into the lungs of several transgenic lines. As expected, the transgenic mice expressed increased amounts of eotaxin mRNA and protein compared with wild-type mice. Surprisingly, following allergen challenge, there was a marked down-regulation of transgene mRNA in three independent transgenic lines. The down-regulation was in contrast to other related proteins such as endogenous eotaxin and surfactant protein D levels, which were both increased following allergen challenge. Consistent with specific down-regulation of the eotaxin transgene, there was no increase in pulmonary eosinophil levels in the transgenic mice above that found in wild-type mice. Analysis of hSP-C transgenic mice with distinct reporter genes and 3'-untranslated regions revealed that allergen challenge was directly affecting the hSP-C promoter. We hypothesized that allergen-induced down-regulation of the hSP-C promoter was related to the eosinophilic inflammation. To test this, we blocked eosinophilic inflammation in the lungs by treating mice with neutralizing antiserum against interleukin-5. Interestingly, this treatment also blocked allergen-induced inhibition of the hSP-C promoter. These results establish that allergic airway inflammation is associated with up-regulation of the surfactant proteins primarily involved in immunity, whereas down-regulation of the surfactant protein primarily involved in maintaining airway patency. Furthermore, the marked down-regulation of the hSP-C promoter is interleukin-5-dependent, implying a critical role for eosinophilic inflammation. These results suggest that alterations in surfactant protein levels may contribute to immune and airway dysfunction in asthma.     

Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12

Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the E. coli dephospho-CoA kinase were identified in a diverse range of organisms.     

Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.     

Molecular cloning, localization and characterization of a 40-kDa catecholamine-regulated protein

We have previously described catecholamine-regulated proteins of molecular masses 47, 40 and 26 kDa (CRP47/40/26). In mammals, these proteins are detected only in brain and have been implicated as playing a role in dopaminergic neurotransmission. In this report, we have cloned the cDNA encoding CRP40 from bovine brain. Analysis of the predicted amino acid sequence revealed that the CRP40 product contains an hsp70 motif and shares homology with heat-shock protein hsp70. Immunolocalization studies using mAbs to dopamine show that it colocalizes with CRP40 in the vesicles of dopaminergic neuroblastoma SH-SY5Y cells. The constitutive expression of CRP40 was increased by exposure to heat shock similar to inducible heat-shock protein hsp70 in SH-SY5Y cells. Dopamine significantly modulated the levels of CRP40, whereas, the expression of hsp70 remained unchanged upon dopamine treatment of these cells. Moreover, CRP40 is able to prevent the thermal aggregation of luciferase in vitro, similar to hsp70, suggesting that CRP40 encodes a dopamine-inducible protein with properties similar to heat-shock proteins. The immunofluorescence analyses show that in SH-SY5Y cells, CRP40 translocates to the nucleus during dopamine-induced apoptosis. These results suggest that CRP40 could play a protective role against the harmful effects of catecholamine metabolites.