IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH.

We present reduced dimensionality (RD) 3D HN(CA)NH for efficient sequential assignment in proteins. The experiment correlates the (15)N and (1)H chemical shift of a residue ('i') with those of its immediate N-terminal (i - 1) and C-terminal (i + 1) neighbors and provides four-dimensional chemical shift correlations rapidly with high resolution. An assignment strategy is presented which combines the correlations observed in this experiment with amino acid type information obtained from 3D CBCA(CO)NH. By classifying the 20 amino acid types into seven distinct categories based on (13)C(ß) chemical shifts, it is observed that a stretch of five sequentially connected residues is sufficient to map uniquely on to the polypeptide for sequence specific resonance assignments. This method is exemplified by application to three different systems: maltose binding protein (42 kDa), intrinsically disordered domain of insulin-like growth factor binding protein-2 and Ubiquitin. Fast data acquisition is demonstrated using longitudinal (1)H relaxation optimization. Overall, 3D HN(CA)NH is a powerful tool for high throughput resonance assignment, in particular for unfolded or intrinsically disordered polypeptides.

High resolution methyl selective ¹³C-NMR of proteins in solution and solid state.

New ¹³C-detected NMR experiments have been devised for molecules in solution and solid state, which provide chemical shift correlations of methyl groups with high resolution, selectivity and sensitivity. The experiments achieve selective methyl detection by exploiting the one bond J-coupling between the ¹³C-methyl nucleus and its directly attached ¹³C spin in a molecule. In proteins such correlations edit the ¹³C-resonances of different methyl containing residues into distinct spectral regions yielding a high resolution spectrum. This has a range of applications as exemplified for different systems such as large proteins, intrinsically disordered polypeptides and proteins with a paramagnetic centre.

Nanopore Sensing of DNA-Histone Complexes on Nucleosome Arrays.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme.

The CRISPR-associated protein 9 (Cas9) system has proven to be a powerful technology for genome editing in a wide variety of in vivo and in vitro applications. CRISPR-Cas9, when loaded with the guide RNA, cleaves the DNA at the target position as recognized by the guide RNA sequence. For successful application of this technology, it is important to study the biophysical parameters affecting its function. Temperature dependence of the Cas9 binding as well as energetics of product release after cleavage has not been well reported in the literature. In this work, we study the binding properties of Cas9 enzyme to the sequence specific target DNA at a range of temperatures and, surprisingly, find that the Cas9 enzyme, in our study, can find and bind its target DNA with 90 ± 20% efficiency at temperatures as low as 4 °C. Further, we show that the cleaved DNA products remain bound to the Cas9 enzyme strongly and is released from the enzyme only at higher temperatures. Using the gel shift assays, we quantify the rate of Cas9 binding to target DNA to be 0.8 ± 0.2 min-1 at 37 °C. We also tested denaturant (SDS) dependent release of cleaved product which showed a similar release pattern with a dissociation constant of 0.23 ± 0.04 mM. Our results of heat and denaturant dependence on Cas9-DNA binding and release mechanics will provide valuable insights for developing temperature dependent applications of the CRISPR-Cas9 technology.

The Viral SUMO-Targeted Ubiquitin Ligase ICP0 is Phosphorylated and Activated by Host Kinase Chk2.

When the herpes simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called Promyelocytic leukemia protein nuclear bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) infected cell polypeptide 0 (ICP0) that degrades PML NBs to alleviate the repression. The molecular details of the mechanism used by ICP0 to target PML NBs are unclear. Here, we identify a bona fide SUMO-interacting motif in ICP0 (SIM-like sequence [SLS] 4) that is essential and sufficient to target SUMOylated proteins in PML NBs such as the PML and Sp100. We shown that phosphorylation of SLS4 creates new salt bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity, and switches ICP0 into a potent STUbL. HSV activates the Ataxia-telangiectasia-mutated kinase-Checkpoint kinase 2 (ATM-Chk2) pathway to regulate the cell cycle of the host. We report that the activated Chk2 also phosphorylates ICP0 at SLS4 and enhances its STUbL activity. Our results uncover that a viral STUbL counters antiviral response by exploiting an unprecedented cross-talk of three post-translational modifications: ubiquitination, SUMOylation, and phosphorylation.

Partial purification and characterization of phytase from Aspergillus foetidus MTCC 11682.

Phytase is a phosphatase enzyme widely used as feed additive to release inorganic phosphorus from plant phytate and enhance its uptake in monogastric animals. Although engineered fungal phytases are used most, a natural enzyme gives opportunity to understand novel properties, if any. In the current study, a novel fungal strain, Aspergillus foetidus MTCC 11682 was immobilized on poly urethane cubes and used for phytase production, purification and molecular characterization. Phytase produced by this method was partially purified by ammonium sulphate precipitation and Sephacryl S-200HR gel filtration to 23.4-fold (compared to crude extract) with recovery of 13% protein. Electrophoresis analysis revealed that phytase has molecular weight of 90.5 kDa on non-reducing and 129.6 kDa on reducing SDS-PAGE. The purified phytase exhibited a wider pH and temperature stability. Analysis of the cloned sequence showed that the gene has 1176 bp that encodes for a peptide of 391 amino acids of the core catalytic region. It was also found that phytase from A. foetidus has a sequence identity of 99% with the phytase gene of other Aspergillus species at nucleotide level and 100% at protein level in A. niger, A. awamori, A. oryzae. In silico analysis of sequence identified the presence of two consecutive and one non-consecutive intra chain disulfide bonds in the phytase. This probably contributed to the differential migration of phytase on reducing and non-reducing SDS-PAGE. There are predicted 11 O-glycosylation sites and 8 N-glycosylation sites, possibly contributed to an enhanced stability of enzyme produced by this organism. This study opened up a new horizon for exploring the novel properties of phytase for other applications.

Efficacy of dietary phytase supplementation on laying performance and expression of osteopontin and calbindin genes in eggshell gland.

This study was conducted to evaluate the effects of different levels of dietary phytase supplementation in the layer feed on egg production performance, egg shell quality and expression of osteopontin (OPN) and calbindin (CALB1) genes. Seventy-five White Leghorn layers at 23 weeks of age were randomly divided into 5 groups consisting of a control diet with 0.33% non-phytate phosphorus (NPP) and 4 low phosphorus (P) diets: 2 diets (T1 and T2) with 0.24% NPP + 250 FTU/kg laboratory produced phytase or commercial phytase and another 2 diets (T3 and T4) with 0.16% NPP + 500 FTU/kg laboratory produced phytase or commercial phytase with complete replacement of inorganic P. The results indicated that there were no significant differences (P > 0.05) in egg production performance and quality of egg during the first 2 months of trial. However, in next 2 months, a significant drop in egg production and feed intake was observed in birds fed diets with low P and 500 FTU/kg supplementation of laboratory produced phytase. Osteopontin gene was up-regulated whereas the CALB1 gene was down regulated in all phytase treatment groups irrespective of the source of phytase. The current data demonstrated that 250 FTU/kg supplementation of laboratory produced phytase with 50% less NPP supplementation and 500 FTU/kg supplementation of commercial phytase even without NPP in diet can maintain the egg production. The up-regulation of OPN and down regulation of CALB1 in egg shell gland in the entire phytase treated group birds irrespective of the source of enzymes is indicative of the changes in P bio-availability at this site.

Effect of immobilized fungal phytase on growth performance and bone traits of broilers fed with low dietary calcium and phosphorus.

AIM: The aim of this study was to investigate the effects of phytase which was laboratory produced by Aspergillus foetidus on the growth performance, mineral retention, and bone traits of broilers fed with low dietary calcium and phosphorus. MATERIALS AND METHODS: The extracellular phytase enzyme secreted into the crude filtrate was concentrated by ammonium sulfate precipitation to obtain an activity of 500 phytase units (FTU). A total of 90 1-day-old chicks (Cobb 500) were randomly divided into three treatment groups with five replicates having six birds each. Dietary treatment, T1, was with 0.45% non-phytate P (NPP) during starter and 0.40% during finisher phase with 1% Ca. Dietary treatment, T2, had 0.37% NPP during starter and 0.32% in finisher phase with 1% Ca and supplemental lab phytase at 500 FTU/kg. Dietary treatment, T3, was similar to T2 with a lower Ca of 0.8%. RESULTS: There was no significant difference among the dietary treatments with regard to body weight gain, feed intake, feed conversion ratio, and Ca retention (p>0.05). However, a significant improvement in retention of P by birds was observed in phytase supplemental groups T2 and T3 (p<0.05). Dry weight of tibia (2.58-2.78 g/kg live weight) and ash content (39.7-41.8%) was comparable among treatments. A similar trend was observed for bone Ca, P, and Mn content. CONCLUSION: The study indicated that 500 FTU/kg phytase can be effectively supplemented in a broiler diet with low phosphorus (0.37% in starter and 0.32% NPP in finisher diet) and low calcium (0.8% in diet) for better growth performance and with successful replacement of dietary P by 0.08 % and reduced P excretion into the environment in broiler chicken.