Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V_max than the full-length form without changes in K_m, which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic α-helix (bold) within amino acid residues 10–24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic α-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.     

How is the relationship between TWIST-1 and BCR-ABL1 gene expressions in chronic myeloid leukaemia patients?

Background: The activation and increased expression of BCR-ABL1 lead to malignant chronic myelogenous leukaemia (CML) cells, as well as the resistance to antitumour agents and apoptosis inducers. Moreover, TWIST-1 protein is a prognostic factor of leukemogenesis, and its level is raised in CML patients with cytogenetic resistance to imatinib. So, there is a likely relationship between BCR-ABL1 and TWIST-1 genes.

Objective: The aim of the study was to assess the relationship between TWIST-1 and BCR-ABL1 expressions.

Methods: Peripheral blood samples were obtained from 44 CML patients under treatment and also from ten healthy subjects as normal controls. The expression of TWIST-1 and BCR-ABL1 genes was measured using real-time PCR, and ABL1 was used as the reference gene. The gene expression was evaluated by REST software.

Results: The expression levels of TWIST-1 and BCR-ABL1 genes in CML patients was changed 40.23?±?177.75-fold and 6?±?18-fold, respectively.

Discussion: No significant relationship was observed between the expressions of TWIST-1 and BCR-ABL1 genes. All patients with TWIST-1 expression levels?≥100-fold had failure of response to treatment.

Conclusion: The probability of the relationship between BCR-ABL1 and TWIST-1 is still debatable, and the average of TWIST-1 expression has been higher in patients without response to treatment. Definitive conclusion needs further investigations.     

Detergency effects of nanofibrillar amyloid formation on glycation of human serum albumin

The prolonged glycation of human serum albumin (HSA) results in significant changes in its structure. The identity of these structural changes and the influence of carbohydrates on these changes require further study. Here, we evaluated structural changes and amyloid formation of HSA upon incubation with Glc, Fru, or Rib. Fluorescence spectrophotometry, surface tension analysis, and transmission electron microscopy (TEM) were utilized to evaluate the structures of glycated HSA. The physicochemical properties including excess free energy, protein adsorption at the air-water interface, critical aggregation concentration (CAC), and surface activity indicated an increase in hydrophobicity and partial unfolding of HSA structure upon glycation. Thus, it appears that AGE products can act as detergents. Incubation of HSA with these sugars after 20 wks induced significant amyloid nanofibril formation. Together these results indicate that prolonged glycation of HSA is associated with a transition from helical structure to beta-sheet (amyloid formation).     

Theoretical study of chemisorption of cyanuric fluoride and S-triazine on the surface of Al-doped graphene

We studied the adsorption of cyanuric fluoride (CF) and s-triazine (ST) molecules on the surface of pristine as well as Al-doped graphenes using density functional theory calculations. Our results reveal low adsorption on the surface of pristine graphene; but by modification of surface using aluminium, resulted Al-doped graphene becomes more reactive towards both CF and ST molecules. We aimed to focus on the adsorption energy, electronic structure, charge analysis, density of state and global indices of each system upon adsorption of CF and ST molecules on the above-mentioned surfaces. Our calculated adsorption energies for the most stable position configurations of CF and ST on Al-doped graphene were ?76.53 kJ mol^?1 (?57.45 kJ mol^?1 BSSE corrected energy) and ?115.55 kJ mol^?1 (?86.87 kJ mol^?1 BSSE corrected energy), respectively, which point to the chemisorption process. For each CF and ST molecule, upon adsorption on the surface of Al-doped graphene, the band gap of HOMO-LUMO was reduced considerably and it becomes a p-type semiconductor, whereas there is no hybridisation between the above-mentioned molecules and pristine graphene.     

Rheumatoid Arthritis Susceptibility Is Associated with the KIR2DS4-Full of Killer-Cell Immunoglobulin-Like Receptor Genes in the Lur Population of Iran

Background:The pathophysiology underlying the progression and development of autoimmune conditions, such as Rheumatoid Arthritis (RA), is a result of dysregulations of the immune system. Research has explored the genetic alterations present in RA; however, limited studies have examined the role of Killer cell Immunoglobulin-like Receptors (KIR) and Human Leukocyte Antigen (HLA) molecules in RA. Therefore, the aim of this study was to examine KIR genes, their HLA ligands, and KIR-HLA compounds in patients with RA.Methods:In this case-control study, a total of 50 patients with RA and 100 healthy individuals were enrolled. DNA samples were evaluated using PCR with sequence specific Primers (PCR-SSP). Odds ratio (OR) with a 95% confidence interval (CI) were reported.Results:Among the KIR genes examined, KIR2DLA (p= 0.0255, OR= 0.389, 95% CI= 0.210-0.722) and KIR2DS4-full (p< 0.0001, OR= 6.163, 95% CI= 3.174-11.968) were observed to have a statistically significant correlation with disease susceptibility to RA. As an inhibitory gene, KIR2DLA was observed to have a protective effect against RA while KIR2DS4-full as an activating gene, was found to increase risk for RA. No significant associations were found between any of the other KIR genotypes, HLA ligands, or KIR-HLA compounds examined in this study to RA susceptibility. Conclusion:In this study of RA in the Lur population of Iran, KIR2DS4-full was observed to increase susceptibility to RA, while KIR2DL5A was found to act as a protecting factor based on both the cross Table and regression analyses. Further research should focus on repeating this study in additional populations.Key Words: HLA, KIR, NK cells, Rheumatoid Arthritis     

The clubroot pathogen Plasmodiophora brassicae: A profile update

Background

Plasmodiophora brassicae is the causal agent of clubroot disease of cruciferous plants and one of the biggest threats to the rapeseed (Brassica napus) and brassica vegetable industry worldwide.

Disease symptoms

In the advanced stages of clubroot disease wilting, stunting, yellowing, and redness are visible in the shoots. However, the typical symptoms of the disease are the presence of club-shaped galls in the roots of susceptible hosts that block the absorption of water and nutrients.

Host range

Members of the family Brassicaceae are the primary host of the pathogen, although some members of the family, such as Bunias orientalis, Coronopus squamatus, and Raphanus sativus, have been identified as being consistently resistant to P. brassicae isolates with variable virulence profile.

Taxonomy

Class: Phytomyxea; Order: Plasmodiophorales; Family: Plasmodiophoraceae; Genus: Plasmodiophora; Species: Plasmodiophora brassicae (Woronin, 1877).

Distribution

Clubroot disease is spread worldwide, with reports from all continents except Antarctica. To date, clubroot disease has been reported in more than 80 countries.

Pathotyping

Based on its virulence on different hosts, P. brassicae is classified into pathotypes or races. Five main pathotyping systems have been developed to understand the relationship between P. brassicae and its hosts. Nowadays, the Canadian clubroot differential is extensively used in Canada and has so far identified 36 different pathotypes based on the response of a set of 13 hosts.

Effectors and resistance

After the identification and characterization of the clubroot pathogen SABATH-type methyltransferase PbBSMT, several other effectors have been characterized. However, no avirulence gene is known, hindering the functional characterization of the five intercellular nucleotide-binding (NB) site leucine-rich-repeat (LRR) receptors (NLRs) clubroot resistance genes validated to date.

Important Link

Canola Council of Canada is constantly updating information about clubroot and P. brassicae as part of their Canola Encyclopedia: https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/ .

Phytosanitary categorization

PLADBR: EPPO A2 list; Annex designation 9E.     

NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.     

The Centrosomal Adaptor TACC3 and the Microtubule Polymerase chTOG Interact via Defined C-terminal Subdomains in an Aurora-A Kinase-independent Manner

The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414–530) and CC2 (aa 530–630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.