Structural and Functional Roles of Coevolved Sites in Proteins

Background

Understanding the residue covariations between multiple positions in protein families is very crucial and can be helpful for designing protein engineering experiments. These simultaneous changes or residue coevolution allow protein to maintain its overall structural-functional integrity while enabling it to acquire specific functional modifications. Despite the significant efforts in the field there is still controversy in terms of the preferable locations of coevolved residues on different regions of protein molecules, the strength of coevolutionary signal and role of coevolution in functional diversification.

Methodology

In this paper we study the scale and nature of residue coevolution in maintaining the overall functionality and structural integrity of proteins. We employed a large scale study to investigate the structural and functional aspects of coevolved residues. We found that the networks representing the coevolutionary residue connections within our dataset are in general of ‘small-world’ type as they have clustering coefficient values higher than random networks and also show smaller mean shortest path lengths similar and/or lower than random and regular networks. We also found that altogether 11% of functionally important sites are coevolved with any other sites. Active sites are found more frequently to coevolve with any other sites (15%) compared to protein (11%) and ligand (9%) binding sites. Metal binding and active sites are also found to be more frequently coevolved with other metal binding and active sites, respectively. Analysis of the coupling between coevolutionary processes and the spatial distribution of coevolved sites reveals that a high fraction of coevolved sites are located close to each other. Moreover, ∼80% of charge compensatory substitutions within coevolved sites are found at very close spatial proximity (< = 5Å), pointing to the possible preservation of salt bridges in evolution.

Conclusion

Our findings show that a noticeable fraction of functionally important sites undergo coevolution and also point towards compensatory substitutions as a probable coevolutionary mechanism within spatially proximal coevolved functional sites.     

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 ^gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.     

Mössbauer and EPR study of iron in vacuoles from fermenting Saccharomyces cerevisiae

Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 μM (57)Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. M?ssbauer spectra of isolated vacuoles were dominated by two spectral features: a mononuclear magnetically isolated high-spin (HS) Fe(III) species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands and superparamagnetic Fe(III) oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a g(ave) ~ 4.3 signal typical of HS Fe(III) with E/D ~ 1/3. Chemical reduction of the HS Fe(III) species was possible, affording a M?ssbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The in vivo vacuolar Fe concentration was estimated to be ~1.2 mM while the Fe concentration of isolated vacuoles was ~220 μM. M?ssbauer analysis of Fe(III) polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, Fe(III) polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. Fe(III) polyphosphate could also be reduced to the Fe(II) state, affording similar M?ssbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the Fe(III) species in vacuoles, but they suggest a complex closely related to Fe(III) polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed.     

Effect of Ayurvedic mercury preparation Makaradhwaja on geriatric canine--a preliminary study

Makaradhwaja, an alchemical Ayurvedic mercury preparation is used as stimulant and vitalizer. Towards veterinary practices, the acceptability, tolerability and toxicity studies were undertaken in geriatric pet dogs aged more than 10 years irrespective of breed and sex for future use. Makaradhwaja (2.5 mg/kg) was used with honey once daily for 30 days. Before and after treatment, blood was collected for hematological studies as well as liver, kidney function and anti-oxidant activity. In control group, honey itself showed no appreciable change whereas, Makaradhwaja lowered neutrophil and total leucocyte count. Serum cholesterol, urea, glucose, alanine amino transferase, aspartate amino transferase, sodium, phosphorus and calcium were decreased. Haemoglobin and serum creatinine were significantly increased. There was appreciable physical, behavioral and body weight change including quality of life. The dose was used in replication of human dose (125 mg/50 kg). Anti-oxidant study showed significant increase of lipid per oxidation in experimental group while the values of ABTS radical cation decolorisation assay although decreased but did not show any significant changes. Decrease of serum urea and increase of serum creatinine could not be explained on single dose response. Different dose study could only explain the optimum dose to be required in canine practices.     

AID binds cooperatively with UNG and Msh2-Msh6 to Ig switch regions dependent upon the AID C terminus

Activation-induced cytidine deaminase (AID) is induced in B cells during an immune response and is essential for both class-switch recombination (CSR) and somatic hypermutation of Ab genes. The C-terminal 10 aa of AID are required for CSR but not for somatic hypermutation, although their role in CSR is unknown. Using retroviral transduction into mouse splenic B cells, we show that the C terminus is not required for switch (S) region double-strand breaks (DSBs) and therefore functions downstream of DSBs. Using chromatin immunoprecipitation, we show that AID binds cooperatively with UNG and the mismatch repair proteins Msh2-Msh6 to Ig Sμ and Sγ3 regions, and this depends on the C terminus and the deaminase activity of AID. We also show that mismatch repair does not contribute to the efficiency of CSR in the absence of the AID C terminus. Although it has been demonstrated that both UNG and Msh2-Msh6 are important for introduction of S region DSBs, our data suggest that the ability of AID to recruit these proteins is important for DSB resolution, perhaps by directing the S region DSBs toward accurate and efficient CSR via nonhomologous end joining.     

Reverse genetics generation of chimeric infectious Junin/Lassa virus is dependent on interaction of homologous glycoprotein stable signal peptide and G2 cytoplasmic domains

The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.     

Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.     

Stimulation of immunity in Indian major carp Catla catla with herbal feed ingredients

Catla catla, catla (150 +/- 20 g) were fed a diet containing seed of Achyranthes aspera (0.5%) and control diet without A. aspera for four weeks prior to and after ip injection with chicken erythrocytes. Fish were sampled for four consecutive weeks after immunization. Hemagglutination antibody titers were significantly higher in the test group of fishes compared with the control group. Serum globulin levels were significantly (P(t-test) < 0.05) higher in the test group than control group on days 14 and 21. Anti-trypsin activity due to total serum protease inhibitors and alpha1-antiprotease was also significantly (P(t-test) < 0.05) higher in the test group of fishes than the control. RNA/DNA ratio of spleen and kidney was also significantly (P(t-test) < 0.05) higher in test group than the control group. All these results confirm that A. aspera enhances the immunity of catla.     

Ochroconis humicola Coexisting with Esthesioneuroblastoma: An Incidental Coloniser or Allergen?

Ochroconis humicola, a fish pathogen, is rarely reported to cause disease in human. We report its first isolation from nasal tissue of a human immunodeficiency virus-positive young female patient. Histopathologically, the nasal mass was diagnosed as esthesioneuroblastoma. She presented with right-sided nasal obstruction and bleeding for two and half months. Computed tomography scan showed the nasal mass filling the whole right nasal cavity, maxillary, ethmoid and sphenoid sinuses. The direct microscopy of the nasal tissue and mucin demonstrated the presence of septate hyphae. On culture, O. humicola was isolated from the same tissue and the fungus was identified by morphologic, physiologic and molecular data including sequencing of ITS and 28S rDNA regions. No antifungal was prescribed, and the whole mass was resected out by endoscopic surgery. The patient was treated further by radical radiotherapy. After 1 year of follow-up, patient is stable with no recurrence of tumour. The role of this fungus was not clear, as it may be bystander or producing allergic fungal rhinosinusitis.