Conformational response to charge clustering in synthetic intrinsically disordered proteins

Background

Recent theoretical and computational studies have shown that the charge content and, most importantly, the linear distribution of opposite charges are major determinants of conformational properties of intrinsically disordered proteins (IDPs). Charge segregation in a sequence can be measured through κ, which represents a normalized measure of charge asymmetry. A strong inverse correlation between κ and radius of gyration has been previously demonstrated for two independent sets of permutated IDP sequences.

Not Only Expansion: Proline Content and Density Also Induce Disordered Protein Conformation Compaction

Intrinsically disordered proteins (IDPs) adopt a wide array of different conformations that can be constrained by the presence of proline residues, which are frequently found in IDPs. To assess the effects of proline, we designed a series of peptides that differ with respect to the number of prolines in the sequence and their organization. Using high-resolution atomistic molecular dynamics simulations, we found that accounting for whether the proline residues are clustered or isolated contributed significantly to explaining deviations in the experimentally-determined gyration radii of IDPs from the values expected based on the Flory scaling-law. By contrast, total proline content makes smaller contribution to explaining the effect of prolines on IDP conformation. Proline residues exhibit opposing effects depending on their organizational pattern in the IDP sequence. Clustered prolines (i.e., prolines with ≤2 intervening non-proline residues) result in expanded peptide conformations whereas isolated prolines (i.e., prolines with >2 intervening non-proline residues) impose compacted conformations. Clustered prolines were estimated to induce an expansion of ∼20% in IDP dimension (via formation of PPII structural elements) whereas isolated prolines were estimated to induce a compaction of ∼10% in IDP dimension (via the formation of backbone turns). This dual role of prolines provides a mechanism for conformational switching that does not rely on the kinetically much slower isomerization of cis proline to the trans form. Bioinformatic analysis demonstrates high populations of both isolated and clustered prolines and implementing them in coarse-grained molecular dynamics models illustrates that they improve the characterization of the conformational ensembles of IDPs.     

Synthetic peptides derived from the C-terminal 6 kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development

Background

Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site.

Methods

In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential.

Results

The final proteolytic step of PfSERA5 involves removal of a C-terminal ~ 6 kDa fragment that results in the generation of a catalytically active ~ 50 kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~ 6 kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5.

Conclusions

Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress.

General significance

These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~ 6 kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.     

Effects of acute and sub-chronic l-dopa therapy on striatal l-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease

Aims

The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study.

Main methods

Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100 mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100 mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined.

Key findings

Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice.

Significance

The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.     

Evidence for the involvement of descending pain-inhibitory mechanisms in the attenuation of cancer pain by carvacrol aided through a docking study

Aims

The present study evaluated the carvacrol (CARV) effect on hyperalgesia and nociception induced by sarcoma 180 (S180) in mice.

Main methods

Carvacrol treatment (12.5–50 mg/kg s.c.) once daily for 15 days was started 24 h after injection of the sarcoma cells in the hind paw (s.c.). Mice were evaluated for mechanical sensitivity (von Frey), spontaneous and palpation-induced nociception, limb use and tumor growth on alternate days. CARV effects on the central nervous system were evaluated through immunofluorescence for Fos protein. Molecular docking studies also were performed to evaluate intermolecular interactions of the carvacrol and muscimol, as ligands of interleukin-10 and GABA_A receptors.

Key findings

CARV was able to significantly reduce mechanical hyperalgesia and spontaneous and palpation-induced nociception, improve use paw, decrease the number of positively marked neurons in lumbar spinal cord and activate periaqueductal gray, nucleus raphe magnus and locus coeruleus. CARV also caused significant decreased tumor growth. Docking studies showed favorable interaction overlay of the CARV with IL-10 and GABA_A.

Significance

Together, these results demonstrated that CARV may be an interesting option for the development of new analgesic drugs for the management of cancer pain.     

Neurotropin® relieves oxaliplatin-induced neuropathy via Gi protein-coupled receptors in the monoaminergic descending pain inhibitory system

Aims

Oxaliplatin is a key drug in the treatment of colorectal cancer, but it causes acute and chronic peripheral neuropathies. We previously reported that repeated administration of neurotropin prevents oxaliplatin-induced mechanical allodynia by inhibiting axonal degeneration in rats. In the present study, we investigated the analgesic effect of a single administration of neurotropin on oxaliplatin-induced neuropathy in rats.

Main methods

Oxaliplatin (4 mg/kg) was administered intraperitoneally twice a week for 4 weeks. Cold hyperalgesia was assessed using the acetone test and mechanical allodynia was evaluated using the von Frey test.

Key findings

Repeated injection of oxaliplatin induced cold hyperalgesia on day 5 and mechanical allodynia on day 28. A single administration of neurotropin transiently relieved both pain behaviors. The analgesic effect of neurotropin was inhibited by pretreatment with 5-HT_1A, 5-HT₂, 5-HT₃, and α2 receptor antagonists and by monoamine depletion. Moreover, the analgesic effect of neurotropin was abolished by intrathecal injection of pertussis toxin, a G_i protein inhibitor.

Significance

These results suggest that neurotropin is effective in relieving oxaliplatin-induced neuropathy, and that G_i protein-coupled receptors in the monoaminergic descending pain inhibitory system may be involved in the analgesic effect of neurotropin. Neurotropin may have clinical potential for the treatment of oxaliplatin-induced neuropathies.     

Time evolution of noise induced oxidation in outer hair cells: Role of NAD(P)H and plasma membrane fluidity

Background

Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation.

Methods

Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy.

Results

In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6 h. Peroxidation occurs after ~ 4 h from noise insult, while ROS are produced in the first 0.2 h and damage cells for a period of time after noise exposure has ended (~ 7.5 h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment.

Conclusions

Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells.

General Significance

Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.     

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride

Background

We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA.

Methods

The combination of experimental and theoretical methods was used.

Results

The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by ³¹P NMR experiments. The thermodynamics of the ligand–nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory.

Conclusions

Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling.

General significance

The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein–DNA interactions. However, these are not clear for the DNA–small molecule interactions and we believe that our studies will bring a new insight into such phenomena.     

Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET

Background

Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.

Methods

In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.

Results

One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (K_a = 4.9 ± 1.9 nM^− 1) with the nanoparticles as compared to a hexahistidine tag (His₆ tag) (K_a ~ 1 nM^− 1).

Conclusion

Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.

General significance

The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.     

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential

Background

Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level.

Methods

MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction.

Results

Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2 × 10⁴ M^- 1 for DNA and 1.06 × 10³ M^− 1for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin.

Conclusions

The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin.

General significance

The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.     

Small-angle X-ray scattering studies of the intact eye lens: Effect of crystallin composition and concentration on microstructure

Background

The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (α, β, and γ). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37 °C) along with several lenses from Antarctic fish (− 2 °C) and subtropical bigeye tuna (18 °C).

Methods

Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure.

Results

We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of α-crystallins.

Conclusions

The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller γ-crystallins in the void space of α-crystallins.

General significance

Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.     

Residue 234 is a master switch of the alternative-substrate activity profile of human and rodent theta class glutathione transferase T1-1

Background

The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site.

Methods

Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants.

Results

The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other.

Conclusions

H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1.

General significance

The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.     

Novel complex Re-Arrangement of ARG1 commonly shared by unrelated patients with Hyperargininemia

Background

Hyperargininemia is a very rare progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. Until now, some mutations were reported worldwide and none of them were of Southeast Asian origins. Furthermore, most reported mutations were point mutations and a few others deletions or insertions.

Objective

This study aims at identifying the disease-causing mutation in the ARG1 gene of Malaysian patients with hyperargininemia.

Methodology

We employed a series of PCR amplifications and direct sequencing in order to identify the mutation. We subsequently used quantitative real-time PCR to determine the copy number of the exons flanking the mutation. We blasted our sequencing data with that of the reference sequence in the NCBI in order to obtain positional insights of the mutation.

Results

We found a novel complex re-arrangement involving insertion, inversion and gross deletion of ARG1 (designated g.insIVS1 + 1899GTTTTATCAT;g.invIVS1 + 1933_ + 1953;g.delIVS1 + 1954_IVS2 + 914;c.del116_188;p.Pro20SerfsX4) commonly shared by 5 patients with hyperargininemia, each originating from different family. None of the affected families share known relationship with each other, although four of the five patients were known to have first-cousin consanguineous parents.

Conclusion

This is the first report of complex re-arrangement in the ARG1. Further analyses showing that the patients have shared the same geographic origin within the northeastern part of Malaysia prompted us to suggest a simple molecular screening of hyperargininemia within related ethnicities using a long-range PCR.     

Enteral calcium infusion used successfully as treatment for a patient with hereditary vitamin D resistant rickets (HVDRR) without alopecia: A novel mutation

Background

We report a novel mutation in a case of hereditary vitamin D resistant rickets (HVDRR) without alopecia and successful management of this condition with the intravenous formulation of calcium chloride delivered via gastric tube.

Clinical Case

A 22 month old male (length − 3.4 SDS; weight − 2.1 SDS) presented with failure to thrive, short stature, severe hypocalcemia and gross motor delay. He did not have alopecia. Initial blood tests and history were thought possibly to suggest vitamin D deficiency rickets: calcium 5.1 mg/dL, (8.8–10.8); phosphorus 4.1 mg/dL, (4.5–5.5); alkaline phosphatase 1481 U/L (80–220); intact PTH 537.1 pg/mL (10–71). Subsequently, vitamin D studies returned that were consistent with HVDRR: 25-hydroxyvitamin D 34 ng/mL (20–100); 1,25-dihydroxyvitamin D 507 pg/mL. This diagnosis was confirmed by DNA sequencing. His subsequent clinical course was complicated by the fact that IV calcium was not a viable option for this patient, and his calcium levels could not be well controlled on oral calcium citrate or calcium glubionate therapy. Eventually, we were able to maintain calcium levels above 8 mg/dL using the intravenous preparation of calcium chloride administered via gastric tube.

Genetic Studies

A unique homozygous T to C base substitution was found in exon 6 in the vitamin D receptor (VDR) gene. This mutation causes leucine to be converted to proline at position 227 in helix 3 in the VDR ligand binding domain (LBD). The mutation rendered the VDR non-functional, leading to HVDRR, with absence of alopecia.

Conclusion

HVDRR should be considered in a patient with profound hypocalcemia which is refractory to conventional therapy of vitamin D deficiency rickets even without evidence of alopecia. We report the first case of HVDRR with a novel mutation in the LBD that was successfully treated with enteral treatment using a calcium chloride infusion.     

Preliminary Investigations on Intradiscal Pressures during Daily Activities: An In Vivo Study Using the Merino Sheep

Purpose

Currently, no studies exist, which attest the suitability of the ovine intervertebral disc as a biomechanical in vivo model for preclinical tests of new therapeutic strategies of the human disc. By measuring the intradiscal pressure in vivo, the current study attempts to characterize an essential biomechanical parameter to provide a more comprehensive physiological understanding of the ovine intervertebral disc.

Methods

Intradiscal pressure (IDP) was measured for 24 hours within the discs L2-L3 and L4-L5 via a piezo-resistive pressure sensor in one merino sheep. The data were divided into an activity and a recovery phase and the corresponding average pressures for both phases were determined. Additionally, IDPs for different static and dynamic activities were analyzed and juxtaposed to human data published previously. After sacrificing the sheep, the forces corresponding to the measured IDPs were examined ex vivo in an axial compression test.

Results

The temporal patterns of IDP where pressure decreased during activity and increased during rest were comparable between humans and sheep. However, large differences were observed for different dynamic activities such as standing up or walking. Here, IDPs averaged 3.73 MPa and 1.60 MPa respectively, approximately two to four times higher in the ovine disc compared to human. These IDPs correspond to lower ex vivo derived axial compressive forces for the ovine disc in comparison to the human disc. For activity and rest, average ovine forces were 130 N and 58 N, compared to human forces of 400-600 N and 100 N, respectively.

Conclusions

In vivo IDPs were found to be higher in the ovine than in the human disc. In contrast, axial forces derived ex vivo were markedly lower in comparison to humans. Both should be considered in future preclinical tests of intradiscal therapies using the sheep. The techniques used in the current study may serve as a protocol for measuring IDP in a variety of large animal models.