Spermine as a possible endogenous allosteric activator of carboxypeptidase A: multispectroscopic and molecular simulation studies

Abstract

Carboxypeptidase A (EC.3.4.17.1) is a zinc-containing proteolytic enzyme that removes the C-terminal amino acid from a peptide chain with the free carboxylate-terminal. In this study, the effect of spermine interaction on the structure and thermal stability of Carboxypeptidase A was investigated by ultraviolet???visible spectroscopy, fluorescence spectroscopy, circular dichroism, Kinetic measurement, molecular docking and simulation studies have also been followed at the pH of 7.5. The transition temperature of Carboxypeptidase A, as a criterion of protein thermal stability, in the presence of spermine was enhanced by increasing the concentration of spermine. The results of fluorescence intensity changes, at two temperatures of 308 and 318?K, also suggested that spermine had a great ability to quench the fluorescence of Carboxypeptidase A through the static quenching procedure. The thermodynamic parameters changes, including standard Gibbs free-energy, entropy and enthalpy, showed that the binding of spermine to Carboxypeptidase A was spontaneous and the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. The changes in the content of the α-helix and the β-sheet of the Carboxypeptidase A with binding to spermine were shown by the CD spectra method. Further, kinetic studies revealed that by increasing concentration of spermine, the activity of Carboxypeptidase A was enhanced. Also, the docking study revealed that the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. As a result, spermine could be considered as an activator and a stabilizer for Carboxypeptidase A.

Communicated by Ramaswamy H. Sarma     

In situ bioremediation of monoaromatic pollutants in groundwater: a review

Monoaromatic pollutants such as benzene, toluene, ethylbenzene and mixture of xylenes are now considered as widespread contaminants of groundwater. In situ bioremediation under natural attenuation or enhanced remediation has been successfully used for removal of organic pollutants, including monoaromatic compounds, from groundwater. Results published indicate that in some sites, intrinsic bioremediation can reduce the monoaromatic compounds content of contaminated water to reach standard levels of potable water. However, engineering bioremediation is faster and more efficient. Also, studies have shown that enhanced anaerobic bioremediation can be applied for many BTEX contaminated groundwaters, as it is simple, applicable and economical.

This paper reviews microbiology and metabolism of monoaromatic biodegradation and in situ bioremediation for BTEX removal from groundwater under aerobic and anaerobic conditions. It also discusses the factors affecting and limiting bioremediation processes and interactions between monoaromatic pollutants and other compounds during the remediation processes.     

Exploring the thermal stability and activity of α-chymotrypsin in the presence of spermine

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.     

Revision of the foraminiferal biozonation scheme in Upper Cretaceous carbonates of the Dezful Embayment,Zagros, Iran: Integrated palaeontological,sedimentological and geochemical investigation

Cretaceous carbonate successions of the Bangestan Group, such as the Sarvak and Ilam formations, are among the most prolific hydrocarbon reserves of the Middle East. However, relatively little is known about their detailed palaeontology and biostratigraphy. Moreover, due to lithological similarity of these carbonate formations recognition of their boundaries in subsurface studies is problematic. To investigate these units, biostratigraphic analyses were carried out on nearly 1100 m of cores, including core plug samples and thin sections prepared from five giant and supergiant oilfields in the northern and southern Dezful Embayment, SW Iran. Accordingly, 59 species of foraminifera (assigned to 43 genera) as well as 11 species of non-foraminifera (10 genera) were recognized. As a result, three biozones were identified, which in stratigraphic order are: Nezzazata-Alveolinids Assemblage Zone; Moncharmontia apenninica-Nezzazatinella-Dicyclina Assemblage Zone; and Rotalia skourensis-algae Assemblage Zone. These are compared with the Wynd's (1965) biozonation scheme, previously introduced in the Zagros area, and a revised scheme is presented. Accordingly, a Cenomanian–Turonian age and a Coniacian–Campanian age are envisaged for the Upper Sarvak and Ilam formations, respectively. In our new biostratigraphic scheme, the Sarvak–Ilam formations boundary is considered to be located above the Moncharmontia apenninica-Nezzazatinella-Dicyclina Assemblage Zone (equivalent of Valvulammina-Dicyclina Assemblage Zone of Wynd, 1965), that is Turonian in age. This zone is bounded by two palaeoexposure surfaces, which correspond approximately to the C–T boundary transitional interval and a post-Turonian, which can be possibly assigned to the Coniacian. Significant sedimentological features of these disconformities include bauxitic–lateritic horizons, karstified profiles and solution-collapsed breccias. Geochemical signatures of these meteorically altered surfaces are also considered to calibrate biofacies and biozones. Finally, we compared our new biozonation scheme with other studies in neighboring areas of SW Iran and the Middle East.     

Optimization of protein extraction from Gelidiella acerosa by carbohydrases using response surface methodology

In this study, application of response surface methodology for enzymic pretreatment optimization of Gelidiella acerosa was investigated in order to improve the extraction of algal proteins using Viscozyme L and Celluclast 1.5L. The total protein, soluble proteins and reducing sugar recovery in the water‐soluble fraction were studied in relation to the hydrolysis time, type and concentration of the enzymes. Enzymatic digestion appeared to be an effective treatment for protein extraction. While enzyme hydrolysis by Celluclast 1.5L was able to facilitate the protein extraction, it was a relatively inefficient way to improve protein extraction yield, in comparison with Viscozyme L. The optimum conditions for protein extraction was found to be hydrolysis by 2.8 μL mL^?1 of Viscozyme L for 12 h.     

The oil palm metallothionein promoter contains a novel AGTTAGG motif conferring its fruit-specific expression and is inducible by abiotic factors

The 1,053-bp promoter of the oil palm metallothionein gene (so-called MSP1) and its 5′ deletions were fused to the GUS reporter gene, and analysed in transiently transformed oil palm tissues. The full length promoter showed sevenfold higher activity in the mesocarp than in leaves and 1.5-fold more activity than the CaMV35S promoter in the mesocarp. The 1,053-bp region containing the 5′ untranslated region (UTR) gave the highest activity in the mesocarp, while the 148-bp region was required for minimal promoter activity. Two positive regulatory regions were identified at nucleotides (nt) −953 to −619 and −420 to −256 regions. Fine-tune deletion of the −619 to −420 nt region led to the identification of a 21-bp negative regulatory sequence in the −598 to −577 nt region, which is involved in mesocarp-specific expression. Gel mobility shift assay revealed a strong interaction of the leaf nuclear extract with the 21-bp region. An AGTTAGG core-sequence within this region was identified as a novel negative regulatory element controlling fruit-specificity of the MSP1 promoter. Abscisic acid (ABA) and copper (Cu²⁺) induced the activity of the promoter and its 5′ deletions more effectively than methyl jasmonate (MeJa) and ethylene. In the mesocarp, the full length promoter showed stronger inducibility in response to ABA and Cu²⁺ than its 5′ deletions, while in leaves, the −420 nt fragment was the most inducible by ABA and Cu²⁺. These results suggest that the MSP1 promoter and its regulatory regions are potentially useful for engineering fruit-specific and inducible gene expression in oil palm.     

Histone deacetylase inhibitors (HDI) cause DNA damage in leukemia cells: a mechanism for leukemia-specific HDI-dependent apoptosis?

Histone deacetylase inhibitors (HDI) increase gene expression through induction of histone acetylation. However, it remains unclear whether increases in specific gene expression events determine the apoptotic response following HDI administration. Herein, we show that a variety of HDI trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses but also actual DNA damage, which is significantly increased in leukemic cells compared with normal cells. Thus, increase in H2AX and ataxia telangiectasia mutated (ATM) phosphorylation, early markers of DNA damage, occurs rapidly following HDI administration. Activation of the DNA damage and repair response following HDI treatment is further emphasized by localizing DNA repair proteins to regions of DNA damage. These events are followed by subsequent apoptosis of neoplastic cells but not normal cells. Our data indicate that induction of apoptosis by HDI may result predominantly through accumulation of excessive DNA damage in leukemia cells, leading to activation of apoptosis.     

Decay-accelerating factor (CD55) is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation

There is compelling evidence that a unique innate immune response in the CNS plays a critical role in host defense and clearance of toxic cell debris. Although complement has been implicated in neuronal impairment, axonal loss, and demyelination, some preliminary evidence suggests that the initial insult consequently activates surrounding cells to signal neuroprotective activities. Using two different models of experimental autoimmune encephalomyelitis, we herein demonstrate selective C1q complement activation on neuron cell bodies and axons. Interestingly, in brains with chronic but not acute experimental autoimmune encephalomyelitis, C3b opsonization of neuronal cell bodies and axons was consistently associated with robust neuronal expression of one of the most effective complement regulators, decay-accelerating factor (CD55). In contrast, levels of other complement inhibitors, complement receptor 1 (CD35), membrane cofactor protein (CD46), and CD59 were largely unaffected on neurons and reactive glial cells in both conditions. In vitro, we found that proinflammatory stimuli (cytokines and sublytic doses of complement) failed to up-regulate CD55 expression on cultured IMR32 neuronal cells. Interestingly, overexpression of GPI-anchored CD55 on IMR32 was capable of modulating raft-associated protein kinase activities without affecting MAPK activities and neuronal apoptosis. Critically, ectopic expression of decay-accelerating factor conferred strong protection of neurons against complement attack (opsonization and lysis). We conclude that increased CD55 expression by neurons may represent a key protective signaling mechanism mobilized by brain cells to withstand complement activation and to survive within an inflammatory site.     

Rapid,reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.