Altered Protein Conformation Research Articles

Deciphering the interaction of bovine heart cystatin with ZnO nanoparticles: Spectroscopic and thermodynamic approach

ZnO-NPs have been widely used in biomedical fields such as therapeutics, cellular imaging, and drug delivery. However, the risk of exposure of nanoparticles to the biological system is not well understood. Nanoparticle-protein interaction is pivotal to understand their biological behavior and predict nanoparticle toxicity that is crucial for its safer applications. In the present study zinc oxide nanoparticles (ZnO-NPs) were synthesized and subjected to interact with buffalo heart cystatin (BHC), purified from buffalo heart, to assess the effect(s) of ZnO-NPs on the structure and function of BHC. In vitro toxicity assessments revealed that BHC, upon interaction with ZnO-NPs, led to the altered protein conformation and perturbed function. A decrease in the anti-papain activity of BHC was observed. Spectroscopic studies demonstrated that formation of BHC-ZnO-NPs complex accompanied by structural changes in BHC along with a significant decrease in its α-helical content. ITC determined the thermodynamic parameters of binding between ZnO-NPs and BHC quantitatively. Increased surface hydrophobicity (change in the tertiary structure) was observed by ANS fluorescence that demonstrated the formation of molten globular intermediates that were found to be stable without any signs of aggregation as depicted by ThT fluorescence. TEM images gave the physical evidence of the formation of ZnO-NPs-BHC corona.

HBsAg mutations related to occult hepatitis B virus infection in HIV-positive patients result in a reduced secretion and conformational changes of HBsAg.

Occult hepatitis B infection (OBI) is a frequent finding in human immunodeficiency virus (HIV)-infected patients. While several related mutations in the hepatitis B virus (HBV) genome have been reported, their distinct impact on HBsAg synthesis is largely obscure. Thirty-one (18%) out of 172 HIV-infected patients, who were selected from HBsAg-negative patients, were positive for HBV-DNA assigned as being OBI-positive. We generated a series of expression constructs of variant HBsAg with "a" determinant amino acid substitutions including P127L, P127T, S136Y, and P127T + S136Y using site-directed mutagenesis. The expression of variant HBsAg was examined by transient transfection in hepatoma cells, followed by HBsAg immunoassay and immunofluorescence stained with specific anti-HBs antibodies. The potential impact of amino acid substitutions at different positions for conformational changes in the HBsAg was investigated using bioinformatics. All variants comprising either single or combined mutations resulted in significantly reduced HBsAg detection in supernatants and in cell lysates of hepatoma cells transfected with the constructs. Moreover, intracellular immunofluorescence staining of cytoblocks showed perinuclear and cytoplasmic fluorescence of HBsAg constructs with significantly diminished fluorescent intensity in comparison to the wild type. Altered protein conformations by predictive models, indicating an impaired detection by the host's immune response as well as by commercial antibody-based test assays. Mutations in the "a" determinant region of HBV as often found in OBI remarkably impair the detection of HBsAg from serum and infected cells, emphasizing the relevance of alternative methods such as HBV-DNA quantification for high-risk groups like HIV-infected individuals. J. Med. Virol. 89:246-256, 2017. © 2016 Wiley Periodicals, Inc.

Photochemical reaction cycle transitions during anion channelrhodopsin gating

A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flash-induced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L⇔M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.

Glycation and secondary conformational changes of human serum albumin: study of the FTIR spectroscopic curve-fitting technique

The aim of this study was attempted to investigate both the glycation kinetics and protein secondary conformational changes of human serum albumin (HSA) after the reaction with ribose. The browning and fluorescence determinations as well as Fourier transform infrared (FTIR) microspectroscopy with a curve-fitting technique were applied. Various concentrations of ribose were incubated over a 12-week period at 37 ± 0.5 ^oC under dark conditions. The results clearly shows that the glycation occurred in HSA-ribose reaction mixtures was markedly increased with the amount of ribose used and incubation time, leading to marked alterations of protein conformation of HSA after FTIR determination.<br /> In addition, the browning intensity of reaction solutions were colored from light to deep brown, as determined by optical observation. The increase in fluorescence intensity from HSA–ribose mixtures seemed to occur more quickly than browning, suggesting that the fluorescence products were produced earlier on in the process than compounds causing browning. Moreover, the predominant α-helical composition of HSA decreased with an increase in ribose concentration and incubation time, whereas total β-structure and random coil composition increased, as determined by curve-fitted FTIR microspectroscopy analysis. We also found that the peak intensity ratios at 1044 cm⁻¹/1542 cm⁻¹markedly decreased prior to 4 weeks of incubation, then almost plateaued, implying that the consumption of ribose in the glycation reaction might have been accelerated over the first 4 weeks of incubation, and gradually decreased. This study first evidences that two unique IR peaks at 1710 cm⁻¹ [carbonyl groups of irreversible products produced by the reaction and deposition of advanced glycation end products (AGEs)] and 1621 cm⁻¹ (aggregated HSA molecules) were clearly observed from the curve-fitted FTIR spectra of HSA-ribose mixtures over the course of incubation time. This study clearly suggests that FTIR spectroscopic curve-fitting technique may be easily used to allow determining the marked changes in the secondary conformational structure and protein aggregation of HSA during ribosylation as well as the production of AGEs.

Binding interaction between rice glutelin and amylose: Hydrophobic interaction and conformational changes

The interaction of rice glutelin (RG) with amylose was characterized by spectroscopic and molecular docking studies. The intrinsic fluorescence of RG increased upon the addition of amylose. The binding sites, binding constant and thermodynamic features indicated that binding process was spontaneous and the main driving force of the interaction was hydrophobic interaction. The surface hydrophobicity of RG decreased with increasing amount of amylose. Furthermore, synchronous fluorescence and circular dichroism (CD) spectra provided data concerning conformational and micro-environmental changes of RG. With the concentration of amylose increasing, the polarity around the tyrosine residues increased while the hydrophobicity decreased. Alteration of protein conformation was observed with increasing of α-helix and reducing of β-sheet. Finally, a visual representation of two binding sites located in the amorphous area of RG was presented by molecular modeling studies.

Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.

Probing the Cl−-pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

Halorhodopsin (HR) functions as a light-driven inward Cl− pump. The Cl− transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl− was added to unphotolyzed Cl−-free NpHR–Brub complex, Brub caused the absorption spectral change in response to the Cl− binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl−-puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl− is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl− already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N–O quasi-equilibrium toward N, supporting Cl− release during the N to O transition. The membrane potential had a much larger effect on the Cl− transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl− transfers during N to O and O to NpHR′ transitions.

Arsenic trioxide binding to serum proteins

Arsenic trioxide (ATO) also known as Trisenox, is an anticancer chemotherapeutic drug which has been used in treating diagnosed and relapsed patients with acute promyelocytic leukemia (APL). Serum albumin is the most abundant of the proteins in blood plasma and is the major transporter for delivering several drugs in vivo. The current study was designed to evaluate the potential ability of human and bovine serum albumin for delivering arsenic trioxide. Therefore, interaction of arsenic trioxide with HSA and BSA was investigated in aqueous solution at physiological conditions using a constant protein concentration and various drug contents. FTIR and UV–Vis spectroscopic methods were used to analyze arsenic trioxide and protein binding modes, the binding constants and the effect of drug complexation on HSA and BSA stability and conformation. Results of this study showed that drug complexation altered protein conformation by major reduction of α-helix and increase of turn structure which is indicative of a partial protein destabilization. Structural analysis revealed that arsenic trioxide bind HSA and BSA with overall binding constants of KATO-HSA=1.07 (±0.01)×104M−1 and KATO-BSA=1.27(±0.02)×104M−1. It could be concluded that serum albumins can be considered as good carriers for delivering arsenic trioxide to target tissue.

Lipid Vesicles for the Skin Delivery of Diclofenac: Cerosomes vs. Other Lipid Suspensions.

Lipid suspensions as drug carriers, including conventional liposomes, ethosomes, transferosomes, proniosomes, niosomes, PEG-PPG-PEG niosomes and stratum corneum liposomes (cerosomes), were formulated and compared. Lipid vesicles were formulated and assessed with regards to enhancement of skin permeation of diclofenac and stability profiles of the formulations. Formulation-induced changes of the biophysical structure of excised human skin were monitored using the Fourier transform infrared spectroscopy. The stability profiles of these suspensions over 12 weeks did not show any significant drug leakage from the vesicles of interest (p > 0.05). FTIR observations indicated that the vesicles increased stratum corneum (SC) lipid fluidization and altered protein conformation. Skin permeability experiments showed that the free unencapsulated drug in the cerosomal formulations caused significant increase in drug permeation across the skin (p < 0.01). Low skin permeability of drug from the other lipid suspensions could be due to the entrapment of diclofenac within these vesicles which decreased the solubility of the hydrophilic drug in the skin lipids and the partition coefficient of the drug from these vesicles into the SC. Optimal drug entrapment in vesicles or alteration of the skin structure may not necessarily enhance the permeation of hydrophilic drugs across the human skin. These lipid vesicles may be further developed into carriers of both hydrophilic and hydrophobic drugs for topical and transdermal delivery, respectively.

Reduced functionality of PSE-like chicken breast meat batter resulting from alterations in protein conformation

The objectives of this study were to evaluate protein thermal stability, water-protein interaction, microstructure, and protein conformation between PSE-like and normal chicken breast meat batters. Sixty pale, soft, and exudative (PSE)-like (L*>53, pH24 h<5.7) and 60 normal (46<L*<53, 5.7<pH24 h<6.1) chicken breast meats were selected from 3 different occasions in a major Chinese commercial plant. Two different meat batters were formulated to 14% meat protein and 2% salt, and they were analyzed for the protein changes and the microstructure using differential scanning calorimetry, low-field (LF)-NMR, SEM, and Raman spectroscopy. PSE-like meat batter had lower gel strength, water-holding capacity, and salt-soluble protein extraction (P < 0.05). Heated PSE-like meat batter formed an aggregated gel matrix, while normal meat batter produced a compact gel network with fine, cross-linked strands by many protein filaments. LF-NMR revealed an increase in the water mobility in heated PSE-like meat batter with an increasing amount of loosely bound water (P < 0.05). No significant changes were observed in the electrophoretic patterns of salt-soluble protein extracts by SDS-PAGE. However, differential scanning calorimetry showed that PSE-like meat had greater myosin and sarcoplasmic proteins/collagen denaturation (P < 0.05). In PSE-like meat, actin denaturation was particular evident after salt addition (P < 0.05) using differential scanning calorimetry. Moreover, Raman spectroscopy indicated that PSE-like meat batter had less unfolded α-helix and β-sheet structure formation, reduced exposure of hydrophobic and tyrosine residues (P < 0.05), and changes in the microenvironment of aliphatic residues and tryptophan, which affected salt-soluble protein extraction, gel properties, and water-holding capacity. In conclusion, the inferior functional properties of PSE-like meat were attributed to not only myosin denaturation, but also actin denaturation after salt addition and different protein structural states.

Effect of Reactive Aldehydes on Ionophore-Mediated Transmembrane Translocations of H+ and K+

Reactive aldehydes (RA) are involved in the onset and progression of many pathologies such as cardiovascular and neurodegenerative diseases. The precise mechanisms by which aldehydes contribute to these diseases remain unclear. Previously, we and other groups suggested that RAs modify the membrane protein function by either binding to the proteins directly(1) or to aminophosphosholipids, with a subsequent alteration in protein conformation. Here, we investigated the influence of biologically important RAs such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-hexenal (HHE) on the transport kinetics of the ionophores carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) and valinomycin (Val). We found that RA increases the membrane conductance, G, in the presence of Val in the following order ONE>HNE>HHE. In contrast G decreases in the presence of CCCP and ONE. The presence of phosphatidylethanolamine in the membrane was crucial for the effect to occur. The results are consistent with the hypothesis that RA adducts alter membrane boundary potential.1. E. A. Malingriaux, et al., “Fatty Acids are Key in 4-Hydroxy-2-Nonenal-Mediated Activation of Uncoupling Proteins 1 and 2” PLoS. ONE. 8(10), e77786 (2013).

From Macroscopic to Molecular Interfaces: How do they Alter Protein Conformation?

Conformation of proteins and peptides are not solely determined by their sequence, but are also strongly dependent on the environmental conditions. In addition to factors like salt concentration or pH, the environment presented by the surrounding molecules also play a major role on the conformational behavior of proteins and peptides. In this study, by using molecular dynamics simulations, we perform a comparative study on the conformational behavior of LKalpha-14 peptides in bulk water vs. macroscopic and molecular interfaces. The molecule under study is designed to have an alpha-helical conformation with the sequence (LKKLLKL)2. Our replica exchange simulations show that, similar to intrinsically disordered proteins, this molecule lacks a unique conformation when isolated in bulk water. However, in the presence of an air/water interface the molecule uniquely adopts the alpha-helix conformation. Further more, such a stabilizing effect is not limited to macroscopic interfaces, but can also be seen in the presence of molecular interfaces presented by surrounding molecules. Even when they are in a disordered state, molecules in solution display temporary molecular interfaces as a result of segregation of their hydrophobic and hydrophilic residues. Our simulations show that, these temporary molecular interfaces act as molecular chaperons in both accelarating the folding of peptides and also stabilize the alpha helical conformation when it forms. In light of these results, LK peptide can be identified as a classic example for conformational selection model.

Spectroscopy and molecular docking studies on the binding of propyl gallate to human serum albumin

The interaction of propyl gallate (PG) with human serum albumin (HSA) was investigated by fluorescence, far-UV CD and FT-IR spectroscopic methods as well as molecular docking. Fluorescence emission spectra demonstrated that the HSA fluorescence was quenched by PG through static quenching and energy transfer with the binding constants in the order of 105Lmol−1. The thermodynamic parameters (ΔH=−29.64KJmol−1, ΔS=2.7Jmol−1K−1) indicated that both hydrophobic force and hydrogen bond interactions played a leading role in the formation of PG–HSA complex. The results also showed the existence of a single binding site, which was located in subdomain IIA (site I) as revealed by molecular docking and competitive binding experiments. Molecular docking studies further showed the participation of several amino acids in PG–HSA complexation, which stabilized by H-bonding systems. The synchronous fluorescence spectra showed that the binding of drug caused the environment of tryptophan residues became more polar. FT-IR and CD spectroscopic further showed that drug complexation altered protein conformation by a major reduction of α-helix inducing a partial protein destabilization.

DETECTION OF PRE-PLAQUE AMYLOID AGGREGATION USING FTIR

Background: Alzheimer's disease (AD) is characterized by misfolding and aggregation of naturally occurring beta-amyloid peptides (Aβ). These aggregates are thought to be pathogenic to neurons, although the conformation of the pathogenic Aβ species remains unclear. Biochemical extraction methods and different microscopy techniques (TEM, confocal) can be used to identify pathogenic Aβ species in the brain, although such methods can alter protein conformation or are n ot designed to determine structural details of protein assemblies.

Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates.

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca+2-regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma.Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

Ligand-Regulated Heterodimerization of Peroxisome Proliferator-Activated Receptor α with Liver X Receptor α

Peroxisome proliferator-activated receptor α (PPARα) and liver X receptor α (LXRα) are members of the nuclear receptor superfamily that function to regulate lipid metabolism. Complex interactions between the LXRα and PPARα pathways exist, including competition for the same heterodimeric partner, retinoid X receptor α (RXRα). Although data have suggested that PPARα and LXRα may interact directly, the role of endogenous ligands in such interactions has not been investigated. Using in vitro protein–protein binding assays, circular dichroism, and co-immunoprecipitation of endogenous proteins, we established that full-length human PPARα and LXRα interact with high affinity, resulting in altered protein conformations. We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPARα ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters. This heterodimer pair was capable of binding to PPARα and LXRα response elements (PPRE and LXRE, respectively), albeit with an affinity lower than that of the respective heterodimers formed with RXRα. LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE. Ectopic expression of PPARα and LXRα in mammalian cells yielded an increased level of PPRE transactivation compared to overexpression of PPARα alone and was largely unaffected by LCFA. Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXRα overexpression alone, and LCFA suppressed transactivation from the LXRE. These data are consistent with the hypothesis that ligand binding regulates heterodimer choice and downstream gene regulation by these nuclear receptors.

Secondary Structure of Proteins is Affected by Complexation with Water Soluble CdSe Quantum Dots

Water-soluble luminescent colloidal quantum dots (QDs) have attracted much attention in biological and medical applications as drug carriers and imaging agents for cells. Investigations of protein–nanoparticle interaction play a key role in biomedical application because when nanoparticles enter the cellular environment, they strongly interact with the intra-cellular protein that may transmit biological signals due to alter protein conformation. The perturbed signal transduction in cells may cause toxicity. Herein, we report on the interaction of semiconductor quantum dots (CdSe) with proteins-bovine serum albumin (BSA), -lactoglobulin ( -Lg) and Human Serum Albumin (HSA). The interactions have been studied by UV-Vis spectroscopy, surface tension measurements, Circular Dichroism (CD) and Raman studies. Protein molecules were adsorbed on the surface of colloidal CdSe through the capping agent 3-mercaptopropionic acid (MPA) making these hydrophilic. Tensiometry and UVabsorbance data showed a finite protein-QDs interaction at different QD concentration. Results from CD and Raman studies suggest that the proteins underwent substantial conformational and hydration changes at the secondary structure level.

Influence of nerve fiber K+ depolarization and altered membrane protein conformation on the state of myelin

Using Raman spectrometry and fluorescence microscopy, we studied the rearrangement of carotenoid molecules and membrane-bound Ca mb 2+ in myelinated nerve fibers after K+ depolarization, K+-channel blocking, and altering the membrane protein conformation. We observed a decrease in Ca mb 2+ and an increase of microviscosity in myelin after depolarization. Changes in Ca mb 2+ and microviscosity were registered after blocking the K+ channels and modifying proteins with PCMB. Our results suggest an interconnection between the condition of nerve fiber membrane proteins, Ca mb 2+ distribution, and myelin microviscosity.

SCA14 mutation V138E leads to partly unfolded PKCγ associated with an exposed C‐terminus, altered kinetics, phosphorylation and enhanced insolubilization

The protein kinase C γ (PKCγ) undergoes multistep activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signalling, such as in spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation owing to an altered protein conformation. As the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14-related PKCγ-V138E exhibits an exposed C-terminus as shown by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster phorbol 12-myristate 13-acetate-induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by coexpressing PDK1 kinase that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C-terminus of the PKCγ-V138E molecule, resulting in a decrease of functional kinase in the soluble fraction. Here, we show that the mutation V138E of the protein kinase C γ (PKCγ) C1B domain (PKCγ-V138E), which is implicated in spinocerebellar ataxia type 14, exhibits a partially unfolded C-terminus. This leads to unusually fast phorbol 12-myristate 13-acetate-induced membrane translocation and accumulation of phosphorylated PKCγ-V138E in the insoluble fraction, causing loss of the functional kinase. In contrast to general chaperones, coexpression of PKCγ's 'natural chaperone', PDK1 kinase, could rescue the PKCγ-V138E phenotype.

Aggregation behavior of casein is correlated with the type of glycation-inducing agent

It has been discovered that protein glycation is involved in several human degenerative diseases and/or disorders, such as diabetes and Alzheimer's disease. Evidence suggests that protein glycation results in the formation of fructosamine and advanced glycation end-products (AGEs), accompanied by the alteration of protein conformation. To investigate how casein is influenced by glycation induced by ribose or methylglyoxal, structural characterization was carried out using thioflavin T (ThT) binding assay, far-UV circular dichroism, intrinsic fluorescence spectroscopy, light scattering, turbidity measurement, and transmission electron microscopy. AGE-related fluorescence measurement and nitro blue tetrazolium assay were used to examine the information regarding the pathway and relevant products associated with glycation. Degree of modification was characterized by determining the contents of lysine and arginine residues. We found that treatment of casein with glycation-inducing agents/glycation modifiers led to an obvious structural unfolding and enhanced solvent-exposed hydrophobic regions. In addition, we found that the concentration profile and production rate of fructosamine and/or AGEs were dependent on the type of glycation-inducing agent used. Moreover, our results showed that amyloid/amyloid-like aggregated species, with relative higher β-sheet secondary structure content and ThT fluorescence-positive characteristics, were found only when casein was treated with methylglyoxal. Finally, modification with methylglyoxal resulted in a higher percentage of reacted lysine or arginine residue in the casein sample. We believe the results of this work could aid in understanding the process of protein glycation.