Studying backbone torsional dynamics of intrinsically disordered proteins using fluorescence depolarization kinetics

Intrinsically disordered proteins (IDPs) do not autonomously adopt a stable unique 3D structure and exist as an ensemble of rapidly interconverting structures. They are characterized by significant conformational plasticity and are associated with several biological functions and dysfunctions. The rapid conformational fluctuation is governed by the backbone segmental dynamics arising due to the dihedral angle fluctuation on the Ramachandran ?–ψ conformational space. We discovered that the intrinsic backbone torsional mobility can be monitored by a sensitive fluorescence readout, namely fluorescence depolarization kinetics, of tryptophan in an archetypal IDP such as α-synuclein. This methodology allows us to map the site-specific torsional mobility in the dihedral space within picosecond-nanosecond time range at a low protein concentration under the native condition. The characteristic timescale of ~?1.4 ns, independent of residue position, represents collective torsional dynamics of dihedral angles (? and ψ) of several residues from tryptophan and is independent of overall global tumbling of the protein. We believe that fluorescence depolarization kinetics methodology will find broad application to study both short-range and long-range correlated motions, internal friction, binding-induced folding, disorder-to-order transition, misfolding and aggregation of IDPs.     

Role of Conserved Cysteines in the Alphavirus E3 Protein

Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.Alphaviruses are members of the Togaviradae family and are single-stranded, positive-sense RNA, enveloped viruses (17). The lipid membranes of the viruses have 80 glycoprotein spikes which are required for viral entry. Each spike is comprised of three copies of a heterodimer which consists of the E2 and E1 proteins (22, 54). E2 and E1 are glycoproteins with a single transmembrane helix that traverses the host-derived lipid bilayer. E2 interacts with the nucleocapsid core at the C terminus (12, 16, 27, 43) and contains the receptor binding site at the N terminus (5, 21, 45). E1 is the viral fusion protein responsible for mediating fusion between the virus membrane and the host cell membrane during an infection (13, 39, 47). Specific interactions in both the ectodomain and transmembrane regions are critical for heterodimer formation (30, 35, 46, 54). The assembly of each heterodimer, its subsequent assembly into a spike, and the interaction of the cytoplasmic tail of the spike with the nucleocapsid core are all essential for the efficient production of infectious particles.Glycoprotein spike assembly requires four structural proteins, E3, E2, 6K, and E1, which are expressed as a single polyprotein. E3 is a small, 64-amino-acid protein (Sindbis virus [SINV] numbering) and contains a signal sequence that translocates the protein into the endoplasmic reticulum (ER) (3, 4, 15). Early in translation, glycosylation of N14 (SINV numbering) occurs and this promotes E3''s release from the ER membrane into the lumen. As a result, the signal sequence is not cleaved from the E3 protein (14). Cellular enzymes cleave the polyprotein to yield pE2 (an uncleaved protein consisting of E3 and E2), 6K, and E1 (23, 55) proteins. In the ER, E1 is found in several conformations, only one of which will form a functional heterodimer with pE2, allowing its transport to the Golgi apparatus (1, 2, 6, 7, 36). After pE2-E1 heterodimerization, self-association between three heterodimers occurs and each individual spike is formed (25, 26, 36). As observed with Semliki Forest virus, disulfide bonds reshuffle within pE2 during protein folding (34), possibly forming intermolecular disulfide bonds between E3 and E2 residues. However, no intermolecular disulfide bonds between pE2 and E1 have been identified (34). Once the viral spikes have been assembled, they are transported to the plasma membrane (11) and are thus exposed to subcellular changes of pH, from pH 7.2 in the ER to pH 5.7 in the vesicles constitutively transporting the spikes to the plasma membrane. In the trans-Golgi network, the E3 protein is cleaved from pE2 by the cellular protein furin (18, 44, 55). E3 remains noncovalently attached to the released virus particle, while in other species E3 is found in the medium of virus-infected cells (32, 49).E3 is required for efficient particle assembly, both in mediating spike folding and in spike activation for viral entry. When an ER signal sequence was substituted for the E3 protein, heterodimerization of pE2 and E1 was abolished (26). Furthermore, when E2 and E1 were expressed individually, low levels of E2 were transported to the cell surface while E1 remained in the ER, suggesting that heterodimerization with pE2 is necessary for E1 to be transported to the cell surface (24, 26, 46). These results are consistent with E3 playing a critical role in mediating the folding of pE2 and the association of pE2 and E1 proteins during spike assembly (7, 38). In viruses where the furin cleavage site was mutated, the virus particles were correctly assembled but severely reduced in infectivity, presumably because the fusion protein was unable to dissociate from pE2 and initiate fusion (44, 55).A comparison of an amino acid sequence alignment of E3 proteins from different alphaviruses (Fig. ​(Fig.1)1) shows that the E3 protein is a small protein with four conserved cysteine (Cys) residues. A subset of E3 proteins contains an additional two Cys residues in a narrow cysteine/proline-rich region, PPCXPCC (Fig. ​(Fig.1).1). We have purified recombinant E3 protein from SINV and have determined that a disulfide bond is present and, furthermore, that these Cys residues are important in virus assembly. Within the alphavirus E3 proteins, we have identified a region that is important for mediating spike transport to the plasma membrane and thus is critical for spike assembly.Open in a separate windowFIG. 1.E3 amino acid sequence alignment from a representative group of alphaviruses. The cysteines marked with asterisks are conserved in all alphavirus species. The ⋄ indicates the conserved but nonessential glycosylation site. The PPCXPCC motif present in ∼50% of alphaviruses is underlined. SFV, Semliki Forest virus; RRV, Ross River virus; BFV, Barmah Forest virus; EEE, eastern equine encephalitis virus; ONN, O''nyong nyong virus; IGB, Igbo Ora virus; OCK, Ockelbo virus; WEE, western equine encephalitis virus; AUR, Aura virus; VEE, Venezuelan equine encephalitis virus.     

Hepatic polyamine metabolism in children with Reye's syndrome.

Acute mitochondrial insult has been suggested as a primary reason for the clinical, histopathological and biochemical abnormalities seen in Reye's syndrome. However, the etiology of mitochondrial dysfunction has not been identified. Polyamines have been known to alter the mitochondrial structure and function. Influenza infection may cause an increase in ornithine decarboxylase activity and thereby channel ornithine for polyamine biosynthesis, leading to mitochondrial dysfunction in Reye's syndrome. To test this hypothesis, the hepatic concentrations of polyamines, polyamine-metabolizing enzymes and urea cycle enzyme activities in Reye's syndrome patients were determined and compared with patients who died from illnesses other than Reye's syndrome. The hepatic concentration of putrescine, spermidine and spermine were increased in Reye's syndrome patients. The activity of ornithine decarboxylase was elevated but, due to the small number of samples, these values did not reach statistical significance. Ornithine carbamoyltransferase activity was decreased in the liver of Reye's syndrome patients. Our results suggest that increased synthesis of polyamines from ornithine may initiate mitochondrial injury in Reye's syndrome.     

Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

Specific oxidized phospholipids (oxPC_CD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPC_CD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPC_CD36 as well as by the strong physiological agonist thrombin. oxPC_CD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPC_CD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPC_CD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPC_CD36.     

Manganese in cell metabolism of higher plants

Manganese, a group VII element of the periodic table, plays an important role in biological systems and exists in a variety of oxidation states. The normal level of Mn in air surrounding major industrial sites is 0.03 μg/m³, in drinking water 0.05 mg/liter and in soil between 560 and 850 ppm. Manganese is an essential trace element for higher plant systems. It is absorbed mainly as divalent Mn²⁺, which competes effectively with Mg²⁺ and strongly depresses its rate of uptake. The accumulation of Mn particularly takes place in peripheral cells of the leaf petiole, petiolule and palisade and spongy parenchyma cells. Mn is involved in photosynthesis and activation of different enzyme systems. Mn deficiency may be expressed as inhibition of cell elongation and yield decrease. Mn toxicity is one of the important growth limiting factors in acid soils. Plant tops are affected to a greater extent than root systems. The toxicity symptoms are, in general, similar to the deficiency symptoms. Toxic effects of Mn on plant growth have been attributed to several physiological and biochemical pathways, although the detailed mechanism is still not very clear. Higher O₂ uptake and loss of control in Mn activated enzyme systems have been associated with Mn toxicity. Mn interferes with the uptake, transport and use of several essential elements including Ca, Fe, Cu, Al, Si, Mg, K, P and N. Excess of Mn reduces the uptake of certain elements and increases that of others. pH plays an important role in Mn uptake. Acidic pH causes a lack of substantial amount of nitrate as an alternative electron acceptor and leads to a high amount of Mn in leaves. High microbial activity, water logging and poorly structured soils cause severe Mn toxicity even in neutral soils. The molecular mechanism of Mn-tolerance is not yet clear. The level of tolerance is different in different species and seems to be controlled by more than one gene. Further information is required on the factors affecting the distribution, accumulation and membrane permeability of the metal in different plant parts and different species. Understanding of the genetic basis of Mn-tolerance is necessary to improve adaptation of crops against acid soils, water logging and other adverse soil conditions.     

Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5 and 3 RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2<20% (putative heterozygotes) in a 1:1 ratio, while the T1 seeds segregated into three classes, C18:2>20%, C18:2 between 12% and 20%) and C18:2<12% (putative homozygotes) in a 1:2:1 ratio. Putative homozygous T1 seeds (C18:2<12% analyzed by the half-seed method) of four of the transgenic lines were grown to establish T2 homozygous lines. These had ca. 73% C18:1 and 8 to 9% each of C18:2 and C18:3 (-linolenic acid) fractions in comparison to ca. 53% C18:1, 24% C18:2 and 16% C18:3 in the parental line VH486.     

Mitochondrial DNA mutations in respiratory complex-I in never-smoker lung cancer patients contribute to lung cancer progression and associated with EGFR gene mutation

Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never‐smoker lung cancer patients including patients with epidermal growth factor receptor (EGFR) and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never‐smoker and 30 current‐smoker lung cancer patients, and determined the mtDNA content. All the patients' samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex‐I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P = 0.006) mtDNA mutation in the never‐smokers compared to the current‐smoker lung cancer patients. MtDNA mutation was significantly higher (P = 0.026) in the never‐smoker Asian compared to the current‐smoker Caucasian patients' population. MtDNA mutation was significantly (P = 0.007) associated with EGFR gene mutation in the never‐smoker patients. We also observed a significant increase (P = 0.037) in mtDNA content among the never‐smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex‐I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion, and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never‐smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never‐smoker lung cancer patients. J. Cell. Physiol. 227: 2451–2460, 2012. © 2011 Wiley Periodicals, Inc.     

Glycodelin A, an immunomodulatory protein in the endometrium, inhibits proliferation and induces apoptosis in monocytic cells

Glycodelin A (GdA), is a lipocalin with an immunomodulatory role, secreted by the endometrium under progesterone regulation and proposed to play a role in protecting the fetus from maternal immune attack. Glycodelin A has an inhibitory effect on the proliferation of T cells and B cells and also on the activity of natural killer cells. We have earlier demonstrated that the inhibitory effect of glycodelin A on T cell proliferation is due to apoptosis induced in these cells through the caspase-dependent intrinsic mitochondrial pathway. Studies reported until now have shown that glycodelin modulates the adaptive immune responses. We, therefore, decided to look at its effect, if any, on the innate immune system. The effect of glycodelin on monocytes was studied using human monocytic cell lines, THP1 and U937, and primary human monocytes as model systems. We demonstrated that glycodelin inhibited the proliferation of THP1 and U937 and induced apoptosis in these cells as well as in primary monocytes. We found that this signaling was caspase-independent but followed the intrinsic mitochondrial pathway of apoptosis. No effect of glycodelin was seen on the phagocytic ability of monocytes post-differentiation into macrophages. These observations suggest that, at the fetomaternal interface, glycodelin plays a protective role by deleting the monocytes that could become pro-inflammatory. Importantly, leaving the macrophages untouched to carry on with efficient clearance of the apoptotic cells.     

Targeted disruption of the mouse Asna1 gene results in embryonic lethality

The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenicals and antimonials. Homologues of the bacterial ArsA ATPase are widespread in nature. We had earlier identified the mouse homologue (Asna1) that exhibits 27% identity to the bacterial ArsA ATPase. To identify the physiological role of the protein, heterozygous Asna1 knockout mice (Asna1+/-) were generated by homologous recombination. The Asna1+/- mice displayed similar phenotype as the wild-type mice. However, early embryonic lethality was observed in homozygous Asna1 knockout embryos, between E3.5 (E=embryonic day) and E8.5 stage. These findings indicate that Asna1 plays a crucial role during early embryonic development.     

Catalytic Nanoceria Are Preferentially Retained in the Rat Retina and Are Not Cytotoxic after Intravitreal Injection

Cerium oxide nanoparticles (nanoceria) possess catalytic and regenerative radical scavenging activities. The ability of nanoceria to maintain cellular redox balance makes them ideal candidates for treatment of retinal diseases whose development is tightly associated with oxidative damage. We have demonstrated that our stable water-dispersed nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in vldlr mutant mice. The objectives of the current study were to determine the temporal and spatial distributions of nanoceria after a single intravitreal injection, and to determine if nanoceria had any toxic effects in healthy rat retinas. Using inductively-coupled plasma mass spectrometry (ICP-MS), we discovered that nanoceria were rapidly taken up by the retina and were preferentially retained in this tissue even after 120 days. We also did not observe any acute or long-term negative effects of nanoceria on retinal function or cytoarchitecture even after this long-term exposure. Because nanoceria are effective at low dosages, nontoxic and are retained in the retina for extended periods, we conclude that nanoceria are promising ophthalmic therapeutics for treating retinal diseases known to involve oxidative stress in their pathogeneses.