Risk analysis of periprosthetic knee joint infection (PJI) in total knee arthroplasty after preoperative corticosteroid injection: a systematic review : A study performed by the Early-Osteoarthritis group of ESSKA-European Knee Associates section.

PURPOSE: Intra-articular corticosteroid injection is widely used for symptomatic relief of knee osteoarthritis. However, if pain is not improved which consequences a total knee arthroplasty (TKA), there is a potential risk of post-operative periprosthetic joint infection (PJI). The aim of this study is to investigate whether the use of preoperative intra-articular corticosteroid injection increases the risk of PJI and to investigate a time frame in which the risk of subsequent infection is significantly increased. METHODS: A systematic search was performed in PubMed (Medline), Scopus, and the Cochrane Library. Inclusion criteria were original studies investigating the rate of PJI in patients receiving pre-operative intra-articular corticosteroid injection compared to controls. RESULTS: A total of 380 unique articles were screened. Six studies met the inclusion criteria with 255,627 patients in total. Overall, no statistical significance was observed in the intra-articular infection rate in corticosteroid compared to controls groups. However, intra-articular corticosteroid injections within 3 months prior to TKA were associated with a significantly increased risk of infection (OR: 1.52, 95% CI 1.37-1.67, p < 0.01); this was not observed in the 6 month period (OR: 1.05, 95% CI 0.80-1.39, p = 0.72). CONCLUSIONS: Performing an intra-articular corticosteroid injection within 3 months prior to TKA is associated with a significantly increased risk of PJI. The current evidence supports the safe use of intra-articular corticosteroid injection more than 6 months before TKA. However, additional studies are needed to clarify the risk of PJI after TKA implantation between 3 and 6 months after the last corticoid injection. LEVEL OF EVIDENCE: IV.

Association between chorionicity and preterm birth in twin pregnancies: a systematic review involving 29 864 twin pregnancies.

BACKGROUND: The perinatal mortality and morbidity among twins vary by chorionicity. Although it is considered that monochorionicity is associated with an increased risk of preterm birth in twin pregnancies, no systematic review exists evaluating this association. OBJECTIVES: This systematic review was undertaken to assess the association between preterm birth and chorionicity in twin pregnancies. SEARCH STRATEGY: We searched the electronic databases from January 1990 to July 2019 without language restrictions. SELECTION CRITERIA: All studies on twin pregnancies where chorionicity and preterm birth were evaluated were included. DATA COLLECTION AND ANALYSIS: Findings are reported as odds ratios with 95% confidence intervals. The estimates are pooled using random-effects meta-analysis. MAIN RESULTS: From 13 156 citations, we included 39 studies (29 864 pregnancies). Monochorionicity was significantly associated with increased risk of preterm birth at ≤28, ≤32, ≤34 and <37 weeks in women asymptomatic and symptomatic for preterm labour (odds ratio [OR] 2.14, 95% CI 1.52-3.02, I2  = 46%, OR 1.55, 95% CI 1.27-1.89 I2  = 68%, OR 1.47, 95% CI 1.27-1.69, I2  = 60%, OR 1.66, 95% CI 1.43-1.93, I2  = 65%, respectively). Among those asymptomatic for preterm labour, significantly increased odds of preterm birth were seen for monochorionicity at gestations ≤34 weeks (OR 1.85, 95% CI 1.42-2.40, I2  = 25%) and <37 weeks (OR 1.75, 95% CI 1.22-2.53, I2  = 61%). Sensitivity analysis showed significantly increased odds of spontaneous preterm birth at ≤34 and <37 weeks for monochorionicity (OR 1.25, 95% CI 1.01-1.55, I2  = 0% and OR 1.41, 95% CI 1.13-1.78, I2  = 0%). CONCLUSIONS: Monochorionicity is significantly associated with preterm birth at all gestations. TWEETABLE ABSTRACT: In twin pregnancies, monochorionicity is associated with an increased risk of preterm birth at all gestations.

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation.

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel, which can be explored by activation of protein ions in a vacuum. Here, we examine the trajectory that Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in a vacuum. We examined wild-type SOD1 and six disease-related point mutants by using tandem MS and ion-mobility MS as a function of collisional activation. For six of the seven SOD1 variants, increasing activation prompted dimers to transition through two unfolding events and dissociate symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition and displayed a much higher abundance of asymmetric products. Supported by the observation that ejected asymmetric G37R monomers were more compact than symmetric G37R ones, we localized this effect to the formation of a gas-phase salt bridge in the first activated conformation. To examine the data quantitatively, we applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals a heightening of the barrier to unfolding in G37R by >5 kJ/mol-1 over the other variants, consistent with expectations for the strength of a salt bridge. Our work demonstrates weaknesses in the simple general framework for understanding protein complex dissociation in a vacuum and highlights the importance of individual residues, their local environment, and specific interactions in governing product formation.

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin.

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core "α-crystallin" domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54-60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54-60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54-60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54-60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.

Evaluating the Effect of Phosphorylation on the Structure and Dynamics of Hsp27 Dimers by Means of Ion Mobility Mass Spectrometry.

The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins.

Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (<1 year to ~17 years death post-diagnosis) that are mutation dependent. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R.

Mass spectrometry data and size exclusion chromatography profiles of Australian taipan venom toxins.

The compositions of paradoxin and taipoxin (PDx and TPx, respectively) were investigated using size exclusion chromatography (SEC) and nano-electrospray ionization mass spectrometry (nano-ESI-MS). The elution profiles from size exclusion chromatography of the venoms from Oxyuranus microlepidotus and Oxyuranus scutellatus were similar. Fractions corresponding to the trimeric toxins were treated with guanidinium hydrochloride and the individual subunits were separated by HPLC. In this report we present the size exclusion chromatography profiles for these toxins, and the nano-ESI mass spectra of the subunits after separation by HPLC: the first such comparative study of these toxins at the protein level. Data in this article are associated with the research article published in Toxicon: "Insight into the subunit arrangement and diversity of paradoxin and taipoxin" (J.A. Harrison, J.A. Aquilina, 2016) [1].

Insights into the subunit arrangement and diversity of paradoxin and taipoxin.

Paradoxin and taipoxin are neurotoxic phospholipases from the inland and coastal species of Australian taipan. Despite their relatively high sequence homology of 70% and 84% for the acidic and basic chains respectively, they differ substantially in reported assays of neurotoxicity. This study provides the first characterisation of paradoxin, which like taipoxin, is a trimer at physiological pH. More broadly, these toxins were found to be composed of a more diverse range of subunits than previously recognised, including newly discovered γTPx isoforms, which give rise to an additional, major conformation of TPx.

Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1.

Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

Age-related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes.

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old-age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA-crystallin peptide (αA(57-65)) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA(57-65) formation escalated significantly. Using 2D gel electrophoresis/nanoLC-ESI-MS/MS, 12 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes.

Stability of the octameric structure affects plasminogen-binding capacity of streptococcal enolase.

Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation.

Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.

Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27.

First-trimester uterine artery Doppler and adverse pregnancy outcome: a meta-analysis involving 55,974 women.

OBJECTIVES: To determine the accuracy with which uterine artery Doppler in the first trimester of pregnancy predicts pre-eclampsia and fetal growth restriction, particularly early-onset disease. METHODS: We searched MEDLINE (1951-2012), EMBASE (1980-2012) and the Cochrane Library (2012) for relevant citations without language restrictions. Two reviewers independently selected studies that evaluated the accuracy of first-trimester uterine artery Doppler to predict adverse pregnancy outcome and performed data extraction to construct 2 × 2 tables. We synthesized sensitivity and specificity for various Doppler indices using a bivariate random-effects model. RESULTS: From 1866 citations, we identified 18 studies (55,974 women). The sensitivity and specificity of abnormal uterine artery flow velocity waveform (FVW) in the prediction of early-onset pre-eclampsia were 47.8% (95% CI: 39.0-56.8) and 92.1% (95% CI: 88.6-94.6), and in the prediction of early-onset fetal growth restriction were 39.2% (95% CI: 26.3-53.8) and 93.1% (95% CI: 90.6-95.0), respectively. The sensitivities for predicting any pre-eclampsia and fetal growth restriction were 26.4% (95% CI: 22.5-30.8) and 15.4% (95% CI: 12.4-18.9), respectively, and the specificities were 93.4% (95% CI: 90.4-95.5%) and 93.3% (95% CI: 90.9-95.1), respectively. The number needed to treat (NNT) with aspirin to prevent one case of early-onset pre-eclampsia fell from 1000 to 173 and from 2500 to 421 for background risks varying between 1% and 0.4%, respectively. CONCLUSIONS: First-trimester uterine artery Doppler is a useful tool for predicting early-onset pre-eclampsia, as well as other adverse pregnancy outcomes. Based on the NNT, abnormal uterine artery Doppler in low-risk women achieves a sufficiently high performance to justify aspirin prophylaxis in those who test positive.

Glutathionylation potentiates benign superoxide dismutase 1 variants to the toxic forms associated with amyotrophic lateral sclerosis.

Dissociation of superoxide dismutase 1 dimers is enhanced by glutathionylation, although the dissociation constants reported to date are imprecise. We have quantified the discreet dissociation constants for wild-type superoxide dismutase 1 and six naturally occurring sequence variants, in their unmodified and glutathionylated forms, at the ratios expressed. Unmodified superoxide dismutase 1 variants that shared similar dissociation constants with SOD1(WT) had disproportionately increased dissociation constants when glutathionylated. This defines a key role for glutathionylation in superoxide dismutase 1 associated familial amyotrophic lateral sclerosis.

Structure-informed design of an enzymatically inactive vaccine component for group A Streptococcus.

UNLABELLED: Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4. IMPORTANCE: We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27.

BACKGROUND: αB-crystallin and HSP27 are mammalian intracellular small heat shock proteins. RESULTS: These proteins exchange subunits in a rapid and temperature-dependent manner. CONCLUSION: This facile subunit exchange suggests that differential expression could be used by the cell to regulate the response to stress. SIGNIFICANCE: A robust technique defines parameters for the dynamic interaction between the major mammalian small heat shock proteins. Small heat shock proteins (sHSPs) exist as large polydisperse species in which there is constant dynamic subunit exchange between oligomeric and dissociated forms. Their primary role in vivo is to bind destabilized proteins and prevent their misfolding and aggregation. αB-Crystallin (αB) and HSP27 are the two most widely distributed and most studied sHSPs in the human body. They are coexpressed in different tissues, where they are known to associate with each other to form hetero-oligomeric complexes. In this study, we aimed to determine how these two sHSPs interact to form hetero-oligomers in vitro and whether, by doing so, there is an increase in their chaperone activity and stability compared with their homo-oligomeric forms. Our results demonstrate that HSP27 and αB formed polydisperse hetero-oligomers in vitro, which had an average molecular mass that was intermediate of each of the homo-oligomers and which were more thermostable than αB, but less so than HSP27. The hetero-oligomer chaperone function was found to be equivalent to that of αB, with each being significantly better in preventing the amorphous aggregation of α-lactalbumin and the amyloid fibril formation of α-synuclein in comparison with HSP27. Using mass spectrometry to monitor subunit exchange over time, we found that HSP27 and αB exchanged subunits 23% faster than the reported rate for HSP27 and αA and almost twice that for αA and αB. This represents the first quantitative evaluation of αB/HSP27 subunit exchange, and the results are discussed in the broader context of regulation of function and cellular proteostasis.

[Organ donation: point of view of private practice physicians in the region Nord-Pas de Calais in France]. / Le don d'organes : point de vue des médecins libéraux de la région Nord-Pas de Calais en France.

BACKGROUND: In 2008, in France, 222 patients died because they did not receive the graft they expected. The main objective of this study was to determine the attitude of private practice physicians concerning organ donation. METHODS: A postal questionnaire was sent to all private practice office-based physicians in the Nord-Pas de Calais region. This questionnaire was elaborated with a panel of physicians and sociologists. It was sent with the monthly journal of the regional union of private physicians of June 2008. RESULTS: Two hundred and seventy eight questionnaires were returned. One hundred and thirty four (48.2%) respondents declared they knew what the regulations about organ donation were. The majority of respondents approved organ donation. Information to patients was provided during visits by 34.53% (96) of practitioners. For those who were knowledgeable about regulations, 50.75% of them talked about organ donation. Physicians who had already had experience with organ donation were more inclined to talk about it with other patients. The practitioners described three actions they felt could have a positive influence on family acceptance: providing information before death, talking about organ donation in an appropriate sensitive way, and relying on the confidence established by a solid patient-physician relationship. CONCLUSION: Almost 90% of private physicians who responded to the survey were in favor of organ donation but only 34.5% delivered information and discussed the issue with their patients. The physicians coped with the topics more easily when they had experienced cases and when they were informed about existing regulations. A specific educational program on the current regulations and how to interact with patients on this subject during a visit could be a first answer to the problem and would be welcomed by practitioners.

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens.

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and ßB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and ßB1-crystallins, suggesting that cross-linking is an alternative pathway for modified ß- and γ-crystallins in the lens.

Understanding the α-crystallin cell membrane conjunction.

PURPOSE: It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS: Fiber cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea, and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. RESULTS: The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an NH2-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. CONCLUSIONS: Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure.

The quaternary organization and dynamics of the molecular chaperone HSP26 are thermally regulated.

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein.