Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach

The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.     

Baicalin protects mice from Staphylococcus aureus pneumonia via inhibition of the cytolytic activity of α-hemolysin

α-Hemolysin (Hla) is a self-assembling, channel-forming toxin that is secreted by Staphylococcus aureus and is central to the pathogenesis of pulmonary, intraperitoneal, intramammary, and corneal infections in animal models. In this study, we report that baicalin (BAI), a natural compound that lacks anti-S. aureus activity, could inhibit the hemolytic activity of Hla. Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that BAI binds to the binding sites of Y148, P151, and F153 in the Hla. This binding interaction inhibits heptamer formation. Furthermore, when added to S. aureus cultures, BAI prevents Hla-mediated human alveolar epithelial (A549) cell injury. In vivo studies further demonstrated that BAI protects mice from S. aureus pneumonia. These findings indicate that BAI hinders the cell lysis activity of Hla through a novel mechanism of interrupting the formation of heptamer, which may lead to the development of novel therapeutics that aim against S. aureus Hla.     

The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-β peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.The proteome is inherently metastable (1, 2), and therefore the cell is required to maintain protein homeostasis (or “proteostasis”) actively through the balancing of a multitude of biochemical pathways (3). The breakdown of this steady state can lead to a variety of diseases, many of which are characterized by the aggregation and deposition of misfolded proteins (4). Molecular chaperones, proteins that act to prevent improper polypeptide associations, are crucial components of the cellular proteostasis machinery (5, 6). They include the small heat-shock proteins (sHSPs), which are found in organisms across all branches of the tree of life and play an important role in preventing protein misfolding and aggregation (7, 8). In general the sHSPs are capable of intercepting destabilized targets (9) and either holding them in a refolding-competent state, preventing them from aggregating into unrecoverable deposits, or directing them toward degradation (10). αB-crystallin (ABC) is an abundant mammalian sHSP, the expression of which is constitutive in most human tissues and up-regulated in a variety of pathological disorders (11). The chaperone activity of ABC has been established for more than two decades (12), and it is associated with amyloid fibril deposits in vivo that are characteristic of protein-misfolding diseases including Alzheimer’s and Parkinson’s diseases (13–15).Proteins enter the amyloid cascade from their native state and form insoluble fibrils via various intermediates, including oligomeric forms (16, 17). Both amyloid fibrils and oligomers are harmful to cells; however, the latter appear to be more toxic (18). ABC has been shown to mitigate amyloid toxicity to cells in culture (19), to interact directly with amyloid oligomers in vitro (20), and to prevent the fibrillation of a variety of targets (21–25). Additionally ABC has been shown to bind to mature amyloid-β peptide (Aβ_1–42) (22, 24), α-synuclein (23, 25), and apolipoprotein C-II (apoC-II) fibrils (26), apparently coating them and preventing their elongation (21). An understanding of how ABC carries out these activities has been hindered by the structural and dynamical complexities of this chaperone. ABC, as is typical for most metazoan sHSPs, consists of a dimeric building block that assembles via terminal interactions into a polydisperse ensemble (8). In ABC, these oligomers range from ∼10–50 subunits and readily interconvert via the exchange of monomers (27) in a process that facilitates the formation of hetero-oligomers between different sHSPs (28). Although several new models for ABC oligomers have been developed recently, a consensus as to their quaternary structure remains to be reached (8, 29).The sequence of ABC can be divided into an Ig-like α-crystallin domain (ACD) that mediates dimerization and is flanked by N- and C-terminal regions that are poorly conserved and variable in length among sHSPs (7, 8). Various regions of the ABC sequence have been implicated as potential binding sites, but there is little consensus (30–34). Moreover, it even remains unclear whether the polydisperse oligomeric ensemble of ABC, the oligomeric dissociation that mediates subunit exchange, or remodeling of the dimer interface is responsible for chaperone function (8). Models have been proposed wherein suboligomeric forms, which are in equilibrium with the assembled state, are the active chaperoning unit. This idea is based on the observation that solution conditions that accelerate subunit exchange also lead to increased chaperone activity (35). Conversely, other studies have demonstrated that ABC still can function as a chaperone despite being cross-linked as an oligomer (36) and that mutations that slow its subunit exchange kinetics do not necessarily diminish its activity as a chaperone in vitro (37).These apparent conflicts likely stem from the intrinsic heterogeneity of ABC and the difficulty in assessing the interplay between its structural and dynamical aspects (8, 29). Here we have gained insight into the chaperone activity of ABC and the prevention of protein aggregation in general by establishing a minimal but chaperone-active unit of ABC that is suitable for structural studies. We drew inspiration from recent structures of the ACD that have revealed the ABC dimer interface to be composed of paired β6+7 strands (38–41). Interestingly, their antiparallel interaction has been crystallized in two different registers, termed “AP_I” and “AP_II” (41), with a third, “AP_III,” having been found for the related HSP20 (38). The three registration states have different amounts of buried surface area in the dimer interface, resulting from monomers progressively being shifted outward relative to one another in AP_II and AP_III compared to AP_I. We have engineered a construct comprising exclusively the core domain of ABC (cABC) which allowed us to test the relationship between oligomeric state, subunit exchange, registration state, and chaperone function of ABC.Our experimental strategy combines X-ray crystallography with native MS and NMR spectroscopy (hereafter, NMR) to examine the structure of cABC. Using a different approach to crystallize the protein, we show that cABC can populate three different registers and that the AP_II form is predominant in solution. Through comparison with a structure of the HSP27 core domain (which we also present here), we have engineered a cysteine mutant of cABC (cABC_E117C) that can be locked into a dimeric state in the AP_II registration state. This protein therefore is unable to exchange monomers under oxidizing conditions. We show that both cABC and cABC_E117C can strongly inhibit amorphous aggregation, amyloidogenesis, and amyloid toxicity as effectively as the wild-type protein. Together our data demonstrate that the core domain of ABC is responsible for its potent molecular chaperone function, which is retained regardless of stoichiometry or registration state of the dimer.     

Characterization of three new deletions at the 5′ end of the HPRT structural gene

Summary In a panel of seven unrelated HPRT-deficient patients three partial deletions of the 5 end of the HPRT structural gene were identified by Southern blot analysis. The deletions could be defined as the loss of exons 1–3, exons 2–3 and exon 3 respectively. In two of the deletion mutations aberrant restriction fragments occurred.Presented at a symposium on inherited metabolic diseases at Brno in 1989.     

Anti–inflammatory activity of the glandular extracts of Thunnus alalunga

ObjectiveTo evaluate the anti-inflamattory activity of Thunnus alalunga by both in vitro and in vivo methods.MethodsAnti-inflammatory activity of the chloroform water extract of Thunnus alalunga was done by both in vitro and in vivo methods. In vitro method was done by human red blood cells membrane stabilization method (HRBC). In vivo evaluation was estimated on Wister albino rats. Acute toxicity studies were done on the extract and no toxicity was reported.ResultsThe percentage protection exhibited by 300 mg/mL concentration was more when compared to the other ones. The 400 mg/mL concentration showed potent activity on comparison with the standard during in vivo evaluation.ConclusionsIn both means of estimation the extract of Thunnus alalunga was found to possess significant anti-inflammatory activity.     

Serum Levels of α1-Antitrypsin Predict Phenotypic Expression of the α1-Antitrypsin Gene

We conducted a retrospective analysis to determine if both ₁-antitrypsin serum level and phenotype need be studied when evaluating children for ₁-AT deficiency. We collected data from patients less than 19 years old who had both serum ₁-AT level and phenotype determined over a 9-year period (January 1992–December 2000). Eighty-eight patients were identified and 15 had the PiZZ phenotype. The serum ₁-AT level was below normal (normal 85–215 mg/dl) in all 15 PiZZ patients. Seventy-two of 73 non-PiZZ patients had normal or above normal serum levels. The sensitivity of the serum ₁-AT level was 100%, and the specificity was 99%. The serum ₁-AT level had a positive predictive value of 94% and a negative predictive value of 100%. We conclude that serum ₁-AT levels are highly predictive of the PiZZ phenotype. Determination of the serum ₁-AT level alone should be the initial test when evaluating for ₁-AT deficiency.     

Antitubercular activity of the semi–polar extractives of Uvaria rufa

ObjectiveTo investigate the inhibitory activity of the chloroform extract, petroleum ether and chloroform sub-extracts, lead-acetate treated chloroform extract, fractions and secondary metabolites of Uvaria rufa (U. rufa) against Mycobacterium tuberculosis (M. tuberculosis) H₃₇Rv.MethodsThe antituberculosis susceptibility assay was carried out using the colorimetric Microplate Alamar blue assay (MABA). In addition, the cytotoxicity of the most active fraction was evaluated using the VERO cell toxicity assay.ResultsThe in vitro inhibitory activity against M. tuberculosis H₃₇Rv increased as purification progressed to fractionation (MIC up to 23 μg/mL). The chloroform extract and its sub-extracts showed moderate toxicity while the most active fraction from chloroform sub-extract exhibited no cytotoxicity against VERO cells. Meanwhile, the lead acetate-treated crude chloroform extract and its fractions showed complete inhibitions (100%) with MIC values up to 8 μg/mL. Phytochemical screening of the most active fraction showed, in general, the presence of terpenoids, steroids and phenolic compounds. Evaluation of the antimycobacterial activity of known secondary metabolites isolated showed no promising inhibitory activity against the test organism.ConclusionsThe present results demonstrate the potential of U. rufa as a phytomedicinal source of compounds that may exhibit promising antituberculosis activity. In addition, elimination of polar pigments revealed enhanced inhibition against M. tuberculosis H₃₇Rv. While several compounds known for this plant did not show antimycobacterial activity, the obtained results are considered sufficient reason for further study to isolate the metabolites from U. rufa responsible for the antitubercular activity.     

Prevalence of α-Thalassemia in the Egyptian Population

Abstract

Hemoglobinopathies are the most common monogenic diseases in the world, causing many health problems worldwide. In Egypt, thalassemia is the most common cause of chronic hemolytic anemia and correlated with significant morbidity and mortality. One thousand Egyptian newborns were screened to detect α-thalassemia (α-thal) deletions using polymerase chain reaction (PCR)-based DNA analysis of cord blood samples. Ninety-one cases (9.1%) of the studied samples were proved to have at least one of the α genes deleted and 851 cases (85.1%) were normal by PCR analysis, while 58 samples (5.8%) failed to be amplified so further DNA analysis could not be done. In the studied group with α gene deletions, we found different types including silent carriers with only one α-globin gene deleted (3.1%), α-thal trait with two α-globin genes deleted (4.2%), Hb H disease with three α-globin genes deleted (1.8%) and no cases carrying Hb Bart’s disease with loss of four α-globin genes. We determined the deletional spectrum of α-thal, which might be used in the future for molecular investigations of the disease in susceptible patients in our population.     

A Newly Identified Deletion of 970 bp at the α-Globin Locus That Removes the Promoter Region of the α1 Gene

The most common causes of α-thalassemia (thal) are deletions that remove a part, or one or both of the functional α-globin genes. These deletions cause diminished expression of the α-globin protein, which may result in relatively low hemoglobin (Hb) and/or mean corpuscular volume (MCV) values. We here report the identification of a 970 bp deletion in the α1-globin gene that encompasses the entire promoter region of the α1-globin gene and 26 bp encoding the 5′ end of the mRNA. Thus, the affected α1-globin gene is prone to be nonfunctional. We therefore nominated the newly identified deletion allele α?α^Δ970. The MCV values of four related carriers of the α?α^Δ970 allele were slightly lowered, consistent with the presence of three functional α-globin genes.     

Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of α-synuclein

Intracellular α-synuclein deposits, known as Lewy bodies, have been linked to a range of neurodegenerative disorders, including Parkinson’s disease. α-Synuclein binds to synthetic and biological lipids, and this interaction has been shown to play a crucial role for both α-synuclein’s native function, including synaptic plasticity, and the initiation of its aggregation. Here, we describe the interplay between the lipid properties and the lipid binding and aggregation propensity of α-synuclein. In particular, we have observed that the binding of α-synuclein to model membranes is much stronger when the latter is in the fluid rather than the gel phase, and that this binding induces a segregation of the lipids into protein-poor and protein-rich populations. In addition, α-synuclein was found to aggregate at detectable rates only when interacting with membranes composed of the most soluble lipids investigated here. Overall, our results show that the chemical properties of lipids determine whether or not the lipids can trigger the aggregation of α-synuclein, thus affecting the balance between functional and aberrant behavior of the protein.The protein α-synuclein is mainly found in the presynaptic termini of neurons (1). The protein has been shown to populate a highly unstructured form in its unbound state both in vitro and in vivo and to adopt an α-helical conformation when bound to membranes (2). The balance between these two states has been found to play a role both in the proposed biological function of the protein, including the regulation of synaptic plasticity, and in the kinetics of its pathogenic aggregation; the latter is the hallmark of a range of diseases, known as synucleinopathies, of which the most common is Parkinson’s disease (3, 4). α-Synuclein has been shown to have its highest affinity for membranes containing either anionic lipids or so-called ”packing defects” (5–7), where the latter are defined as low-density regions in bilayers with high exposure of the lipid hydrophobic chains attributable to a mismatch between lipid shape and bilayer curvature (6, 7).Biological membranes are highly heterogeneous and differ from one cell or organelle to another in terms of the physical and chemical properties of the membranes, including curvature, charge, fluidity, and packing of the hydrophobic chains (8–10). The variety of membrane structures in cells can be directly related to differences in lipid (and protein) composition, where properties such as length and saturation of the hydrocarbon chain as well as the charge and size of the polar head group are crucial in determining the properties of the membrane (8, 9). In particular, most chemical and thermotropic properties of a lipid molecule are known to vary almost linearly with the length of its hydrophobic chain. As some examples, the standard change in free energy of transfer of a lipid molecule from water into a bilayer (i.e., its solubility in water), the melting temperature, and the enthalpy of melting have all been found to be proportional to the number of aliphatic carbons in the hydrophobic chain, which ranges from 8 to 18 (11). In addition, the adsorption and partitioning of small molecules and proteins to membranes can also affect the structural and thermotropic properties of the latter, and the magnitude and characteristics of these changes depend on the nature of the molecular interactions (e.g., electrostatic, hydrophobic) (12, 13).The interactions between amphipathic proteins and membranes have been extensively studied over the last three decades (7, 14–22). In general, the amino acid sequences of these peripheral proteins are characterized by patterns of hydrophobic and polar residues such that the proteins fold into amphipathic α-helices upon binding to hydrophobic patches exposed at the membrane interface (16, 17). In particular, molecular dynamics simulations and neutron reflectometry studies of deposited bilayers have shown that the amphipathic helix in α-synuclein is primarily located in the vicinity of the lipid phosphate groups and the glycerol backbone (16, 23–25).Although the binding of α-synuclein to membranes has been well characterized for different lipid systems (26–28), the observed modulation of the kinetics of the conversion of monomeric α-synuclein into amyloid fibrils by different membranes is less well understood (29–32). Most studies of this phenomenon have been performed under conditions of mechanical agitation (32) and/or in the presence of catalyzing polymer surfaces (31), where α-synuclein aggregates also in the absence of lipids and where the mechanism of aggregation has not yet been elucidated. Here, we take a different approach using an experimental procedure with protein-repellant surfaces and under quiescent conditions (33) that enables the systematic study of the manner in which a change in lipid properties can affect the ability of a model membrane to initiate α-synuclein aggregation. Indeed, we have previously shown that the presence of model membranes composed of 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) triggers the aggregation of α-synuclein by specifically enhancing the rate of primary nucleation (33). In addition, this study showed how the protein:lipid (P:L) ratio modulates the kinetics of α-synuclein aggregation in the presence of DMPS; at low P:L ratios, effectively all of the protein molecules are adsorbed onto the surface of the membrane in a thermodynamically stable α-helical state and no aggregation is observed. At high P:L ratios, however, the protein molecules populate both the free monomeric state and the membrane-bound state, leading to rapid amyloid formation (33).In the present study, we have applied this experimental procedure to probe how changes in the chemical (charge and solubility) and physical (thermotropic) properties of lipids affect the binding of α-synuclein and the magnitude by which model membranes can trigger α-synuclein aggregation. The results reveal that the efficiency of the binding of α-synuclein to model membranes is correlated with their fluidity and, conversely, that the self-assembly of the lipids is affected by their association with the protein. In addition, although α-synuclein has a high affinity for all of the fluid anionic model membranes investigated here, this interaction is not sufficient for the efficient induction of aggregation. Rather, the rate of amyloid fibril formation is shown to be inversely correlated with the free energy of transfer of the lipid molecule from water into the bilayer. These results indicate that the chemical properties of the lipids are likely to play an important role in perturbing the balance between functional and deleterious interactions of α-synuclein with membranes.     

Association of physical activity and sedentary time with structural brain networks—The Maastricht Study

We assessed whether objectively measured low- and high-intensity physical activity (LPA and HPA) and sedentary time (ST) were associated with white matter connectivity, both throughout the whole brain and in brain regions involved in motor function. In the large population-based Maastricht Study (n = 1715, age 59.6 ± 8.1 (mean ± standard deviation) years, and 48% women), the amounts of LPA, HPA, and ST were objectively measured during 7 days by an activPAL accelerometer. In addition, using 3T structural and diffusion MRI, we calculated whole brain node degree and node degree of the basal ganglia and primary motor cortex. Multivariable linear regression analysis was performed, and we report standardized regression coefficients (stβ) adjusted for age, sex, education level, wake time, diabetes status, BMI, office systolic blood pressure, antihypertensive medication, total-cholesterol-to-HDL-cholesterol ratio, lipid-modifying medication, alcohol use, smoking status, and history of cardiovascular disease. Lower HPA was associated with lower whole brain node degree after full adjustment (stβ [95%CI] = − 0.062 [− 0.101, − 0.013]; p = 0.014), whereas lower LPA (stβ [95%CI] = − 0.013 [− 0.061, 0.034]; p = 0.580) and higher ST (stβ [95%CI] = − 0.030 [− 0.081, 0.021]; p = 0.250) was not. In addition, lower HPA was associated with lower node degree of the basal ganglia after full adjustment (stβ [95%CI] = − 0.070 [− 0.121, − 0.018]; p = 0.009). Objectively measured lower HPA, but not lower LPA and higher ST, was associated with lower whole brain node degree and node degree in specific brain regions highly specialized in motor function. Further research is needed to establish whether more HPA may preserve structural brain connectivity.

     

Effect of thermal cutaneous stimulation on the gastric motor activity：Study of the mechanism of action

AIM：To investigate the mechanism of action of thermal cutaneous stimulation on the gastric motor inhibition. METHODS：The gastric tone of 33 healthy volunteers （20 men, mean age 36.7 ± 8.4 years） was assessed by a barostat system consisting of a balloon-ended tube connected to a strain gauge and air-injection system. The tube was introduced into the stomach and the balloon was inflated with 300 mL of air. The skin temperature was elevated in increments of 3℃ up to 49℃ and the gastric tone was simultaneously assessed by recording the balloon volume variations expressed as the percentage change from the baseline volume. The test was repeated after separate anesthetization of the skin and stomach with lidocaine and after using normal saline instead of lidocaine. RESULTS：Thermal cutaneous stimulation resulted in a significant decrease of gastric tone 61.2% ± 10.3% of the mean baseline volume. Mean latency was 25.6 ± 1.2 ms. After 20 min of individual anesthetization of the skin and stomach, thermal cutaneous stimulation produced no significant change in gastric tone. CONCLUSION：Decrease in the gastric tone in response to thermal cutaneous stimulation suggests a reflex relationship which was absent on individual anesthetization of the 2 possible arms of the reflex arc：the skin and the stomach. We call this relationship the＂cutaneo-gastric inhibitory reflex＂. This reflex may have the potential to serve as an investigative tool in the diagnosis of gastric motor disorders, provided further studies are performed in this respect.     

Molecular Characterization of α-Thalassemia in the Dohuk Region of Iraq

The molecular basis of α-thalassemia (α-thal) has been addressed by several studies from the eastern Mediterranean region, but not from Iraq. To address this issue, we studied 51 individuals with unexplained hypochromia and/or microcytosis, as well as nine patients with documented Hb H disease from the Dohuk region in northern Iraq. We used multiplex gap-polymerase chain reaction (gap-PCR), reverse hybridization, and sequencing for this purpose. It was found that the most common genotypes in those with unexplained hypochromia and/or microcytosis were ?α^3.7/αα, followed by ? ?^MED-I/αα, then ?α^3.7/?α ^3.7, respectively, detected in 84.3% of the above individuals. Other genotypes identified sporadically were ?α^4.2/αα, α^{poly A1}α/αα (AATAAA>AATAAG), α^Adanaα/αα [Hb Adana, codon 59 (Gly→Asp) or HBA1:c.179G>A], and α^Evanstonα/αα [Hb Evanston, codon 14 (Trp→Arg) or HBA1:c.43 T>C]. Three cases (5.88%) remained uncharacterized even after sequencing. All nine Hb H cases carried the ?α^3.7/? ?^MED-I genotype. Such findings are rather different from those in other eastern Mediterranean populations, particularly with relevance to an Hb H molecular basis.     

Reprint of: the paradox of α-adrenergic coronary vasoconstriction revisited

Activation of coronary vascular α-adrenoceptors results in vasoconstriction which competes with metabolic vasodilation during sympathetic activation. Epicardial conduit vessel constriction is largely mediated by α(1)-adrenoceptors; the constriction of the resistive microcirculation largely by α(2)-adrenoceptors, but also by α(1)-adrenoceptors. There is no firm evidence that α-adrenergic coronary vasoconstriction exerts a beneficial effect on transmural blood flow distribution. In fact, α-blockade in anesthetized and conscious dogs improves blood flow to all transmural layers, during normoperfusion and hypoperfusion. Also, in patients with coronary artery disease, blockade of α(1)- and α(2)-adrenoceptors improves coronary blood flow, myocardial function and metabolism. This article is part of a Special Issue entitled "Coronary Blood Flow".