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The glycation and aggregation of lens proteins significantly contribute to the onset of diabetic cataracts as well as the retinopathy. The glycation exerts numerous alterations in the tertiary structural of proteins. Moreover, the covalent crosslinking of lens crystallins also contribute to the cataract formation. In this article, the effect of pioglitazone on glucose induced glycation and aggregation α-crystallin was examined. A remarkable inhibition of early glycation products (~80%) and advanced glycation products (~75%) was recorded by the treatment of pioglitazone. There was75% recovery in biochemical marker (carbonyl content). The presence of 150 μM of pioglitazone reduced the free lysine modifications to 35%. Treatment of pioglitazone also protected the secondary structural alterations induced by glycation and inhibited the formation of protein aggregates. The interaction studies showed that pioglitazone interacted with α-crystallin via moderate binding affinity. The interaction between pioglitazone interacted and α-crystallin was energetically and entropically favourable. The complex of pioglitazone with studied protein stable in which RMSF, R

Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.

PURPOSE: This research aims to probe the interaction of α-crystallin with a model of human, porcine, and mouse lens-lipid membranes. METHODS: Cholesterol/model of human lens-lipid (Chol/MHLL), cholesterol/model of porcine lens-lipid (Chol/MPLL), and cholesterol/model of mouse lens-lipid (Chol/MMLL) membranes with 0 to 60 mol% Chol were prepared using the rapid solvent exchange method and probe-tip sonication. The hydrophobicity near the surface of model lens-lipid membranes and α-crystallin association with these membranes were investigated using the electron paramagnetic resonance spin-labeling approach. RESULTS: With increased Chol content, the hydrophobicity near the surface of Chol/MHLL, Chol/MPLL, and Chol/MMLL membranes, the maximum percentage of membrane surface occupied (MMSO) by α-crystallin, and the association constant (K(a)) decreased, showing that surface hydrophobicity of model lens-lipid membranes modulated the α-crystallin association with these membranes. The different MMSO and K(a) for different model lens-lipid membranes with different rates of decrease of MMSO and K(a) with increased Chol content and decreased hydrophobicity near the surface of these membranes suggested that the lipid composition also modulates α-crystallin association with membranes. Despite different lipid compositions, complete inhibition of α-crystallin association with model lens-lipid membranes was observed at saturating Chol content forming cholesterol bilayer domains (CBDs) with the lowest hydrophobicity near the surface of these membranes. The decreased mobility parameter with increased α-crystallin concentration suggested that membranes near the surface became less mobile due to α-crystallin association. The decreased mobility parameter and increased maximum splitting with increased Chol content suggested that membranes became less mobile and more ordered near the surface with increased Chol content. CONCLUSIONS: This study suggested that the interaction of α-crystallin with model lens-lipid membranes is hydrophobic. Furthermore, our data indicated that Chol and CBDs reduce α-crystallin association with lens membrane, likely increase α-crystallin concentration in lens cytoplasm, and possibly favor the chaperone-like activity of α-crystallin maintaining lens cytoplasm homeostasis.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

The absence of cellular organelles in fiber cells and very high cytoplasmic protein concentration (up to 900 mg/ml) minimize light scattering in the lens and ensure its transparency. Low oxygen concentration, powerful defense systems (antioxidants, antioxidant enzymes, chaperone-like protein alpha-crystallin, etc.) maintain lens transparency. On the other hand, the ability of crystallins to accumulate age-associated post-translational modifications, which reduce the resistance of lens proteins to oxidative stress, is an important factor contributing to the cataract formation. Here, we suggest a mechanism of cataractogenesis common for the action of different cataractogenic factors, such as age, radiation, ultraviolet light, diabetes, etc. Exposure to these factors leads to the damage and death of lens epithelium, which allows oxygen to penetrate into the lens through the gaps in the epithelial layer and cause oxidative damage to crystallins, resulting in protein denaturation, aggregation, and formation of multilamellar bodies (the main cause of lens opacification). The review discusses various approaches to the inhibition of lens opacification (cataract development), in particular, a combined use of antioxidants and compounds enhancing the chaperone-like properties of alpha-crystallin. We also discuss the paradox of high efficiency of anti-cataract drugs in laboratory settings with the lack of their clinical effect, which might be due to the late use of the drugs at the stage, when the opacification has already formed. A probable solution to this situation will be development of new diagnostic methods that will allow to predict the emergence of cataract long before the manifestation of its clinical signs and to start early preventive treatment.

Small heat shock proteins (sHsps) containing conserved α-crystallin domain play important roles in many cellular processes, but little is known about the functions of sHsps in filamentous entomopathogens. Here, three sHsps of Hsp20, Hsp30a, and Hsp30b were characterized in

Alpha-crystallin (α-crystallin) is an important eye protein having chaperone activity; its aggregation and precipitation are vital in cataract development. Polyethylene glycol-400 (PEG-400) is an i...

Cataract, the major cause of blindness worldwide occurs due to the misfolding and aggregation of the protein crystallin, which constitute a major portion of the lens protein. Other than the whole protein crystallin, the peptide sequences generated from crystallin as a result of covalent protein damage have also been shown to possess and foster protein aggregation, which can be established as crystallin aggregation models. Thus, the disaggregation or inhibition of these protein aggregates could be a viable approach to combat cataract and preserve lens proteostasis. Herein, we tried to explore the disruption as well as inhibition of the intact α-crystallin protein and α-crystallin derived model peptide aggregates by l-3,4-dihydroxyphenylalanine (levodopa) coated gold (Au) nano/micro-roses as modulators. Thioflavin T fluorescence enhancement assay, and electron microscopic analysis were being employed to probe the anti-aggregation behavior of the Au nano/micro-roses towards the aggregating α-crystallin peptides/protein. Further, computational studies were performed to reveal the nature of molecular interactions between the levodopa molecule and the α-crystallin derived model peptides. Interestingly, both levodopa coated Au nano/micro-roses were found to be capable of inhibiting as well as preventing the aggregation of the intact α-crystallin protein and other model peptides derived from it.

In lens, ∼90% of ocular proteins are αβγ-crystallins with concentrations ≥400 mg/ml, which need to remain soluble for the whole life-span and their aggregation leads to cataract. The G18V mutation of human γS-crystallin causes hereditary childhood-onset cortical cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3–7 mM. Very recently, we found that ATP has no significant binding to γS-crystallin as well as no alternation of its conformation. Nevertheless, ATP antagonizes the crowding-induced destabilization of γS-crystallin even at 1:1, most likely by interacting with the hydration shell. Here by DSF and NMR, we characterized the effect of ATP on binding, conformation, stability of G18V γS-crystallin and its interactions with α-crystallin. The results reveal: 1) G18V significantly accelerates the crowding-induced destabilization with Tm of 67 °C reduced to 50.5 °C at 1 mM. 2) Most unexpectedly, G18V almost completely eliminates the antagonizing effect of ATP against the crowding-induced destabilization. 3) ATP shows no significant effect on the interactions of α-crystallin with both WT and G18V γS-crystallin. Results together decode for the first time that G18V causes cataract not only by accelerating the crowding-induced destabilization, but also by eliminating the antagonizing effect of ATP against the crowding-induced destabilization.

Background: Loss of a-crystallin chaperone function results in the lens protein aggregation leading to cataract. In this study, we evaluated the efficacy of micellar lutein with different fatty acids in modulating a-crystallin chaperone function under selenite cataract conditions. Methods: Cataract was induced in rat pups by giving sodium selenite (25 mM/kg body weight) by IP. Lutein [(L), 1.3 mmol/kg body weight)] was given day before and five days after selenite injection as a micelle with 7.5 mM linoleic acid (LA), or 7.5 mM eicosapentaenoic acid (EPA)þdocosahexaenoic acid (DHA) or 7.5 mM oleic acid (OA). Lens a-crystallins was purified, and its chaperone function and integrity was assessed. Cholesterol, calcium, calpain-2, procaspase-3, and expression of a-A and b-B1 crystallin in the lens of cataract and micellar lutein administered rats were evaluated. Results: Cataract induction significantly (p < 0.05) decreased lens a-crystallin chaperone function. Cataract rats had increased cholesterol and calcium level, increased the expression of calpain-2, and a-A and b-B1 crystallin, and reduced the pro-caspase-3 level in the lens. However, micellar lutein administration significantly (p < 0.05) protected client proteins from aggregation via the modulation of calciumdependent calpain-2 protease activity. The chaperone function of lens a-crystallins in rats administered micellar lutein with EPA þ DHA was found to be highest when compared to OA and LA. Conclusions: Micellar lutein with unsaturated fatty acids beneficially modulates a-crystallin chaperone function. Among the fatty acids tested, micellar lutein with EPA þ DHA exhibited superior effects, thereby offering a promising strategy for cataract management.

PURPOSE/AIM: The amount of membrane-bound α-crystallin increases significantly with age and cataract formation, accompanied by a corresponding decline in the level of α-crystallin in the lens cytoplasm. The purpose of this research is to evaluate the binding affinity of α-crystallin to the phospholipid membranes as well as the physical properties of the membranes after α-crystallin binding. MATERIALS AND METHODS: The continuous wave and saturation recovery electron paramagnetic resonance (EPR) methods were used to obtain the information about the binding affinity and the physical properties of the membrane. In this approach, the cholesterol analogue spin label CSL was incorporated in the membrane and the binding of α-crystallin to the membrane was monitored by this spin label. Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with 1% of CSL. The measured membrane properties included the mobility parameter, fluidity and the oxygen transport parameter. RESULTS: The binding affinity (K(a)) of α-crystallin with the POPC membrane was estimated to be 4.9 ± 2.4 μM(−1). The profiles of mobility parameter showed that mobility parameter decreased with an increase in the binding of α-crystallin. The profiles of spin-lattice relaxation rate showed that the spin-lattice relaxation rate decreased with an increase in binding. These results show that the binding of α-crystallin makes the membrane more immobilized near the head group region of the phospholipids. Furthermore, the profiles of the oxygen transport parameter indicated that the oxygen transport parameter decreased with an increase of binding, indicating the binding of α-crystallin forms a barrier for the passage of non-polar molecules which supports the barrier hypothesis. CONCLUSIONS: The binding of α-crystallin to the membrane alters the physical properties of the membranes, and this plays a significant role in modulating the integrity of the membranes. EPR techniques are useful in studying α-crystallin membrane interactions.

Alzheimer's disease (AD) is a chronic and irreversible neurodegenerative disorder. Abnormal aggregation of the neurotoxic amyloid‑β (Aβ) peptide is an early event in AD. The activation of astrocytic α7 nicotinic acetylcholine receptor (α7 nAChR) can inhibit Aβ aggregation; thus, the molecular mechanism between α7 nAChR activation and Aβ aggregation warrants further investigation. In the present study, Aβ oligomer levels were assessed in astrocytic cell lysates after treatment with PNU282987 (a potent agonist of α7 nAChRs) or co‑treatment with LY294002, a p‑Akt inhibitor. The levels of heat shock factor‑1 (HSF‑1), heat shock protein 70 (HSP‑70), and αB‑crystallin (Cryab) in astrocytes treated with PNU282987 at various time‑points or co‑treated with methyllycaconitine (MLA), a selective α7 nAChR antagonist, as well as co‑incubated with LY294002 were determined by western blotting. HSP‑70 and Cryab levels were determined after HSF‑1 knockdown (KD) in astrocytes. PNU282987 markedly inhibited Aβ aggregation and upregulated HSF‑1, Cryab, and HSP‑70 in primary astrocytes, while the PNU282987‑mediated neuroprotective effect was reversed by pre‑treatment with MLA or LY294002. Moreover, the HSF‑1 KD in astrocytes effectively decreased Cryab, but not HSP‑70 expression. HSF‑1 is necessary for the upregulation of Cryab expression, but not for that of HSP‑70. HSF‑1 and HSP‑70 have a neuroprotective effect. Furthermore, the neuroprotective effect of PNU282987 against Aβ aggregation was mediated by the canonical PI3K/Akt signaling pathway activation.

Distal hereditary motor neuropathies (dHMN) are a group of inherited peripheral nerve disorders characterized by length-dependent motor neuron weakness and subsequent muscle atrophy. Missense mutations in the gene encoding small heat shock protein HSPB1 (HSP27) have been associated with hereditary neuropathies including dHMN. HSPB1 is a member of the small heat shock protein (sHSP) family characterized by a highly conserved α-crystallin domain that is critical to their chaperone activity. In this study, we modeled HSPB1 mutant-induced neuropathies in Drosophila using a human HSPB1S135F mutant that has a missense mutation in its α-crystallin domain. Overexpression of the HSPB1 mutant produced no significant defect in the Drosophila development, however, a partial reduction in the life span was observed. Further, the HSPB1 mutant gene induced an obvious loss of motor activity when expressed in Drosophila neurons. Moreover, suppression of histone deacetylase 6 (HDAC6) expression, which has critical roles in HSPB1 mutant-induced axonal defects, successfully rescued the motor defects in the HSPB1 mutant Drosophila model.

Cataract induced by exposure to naphthalene is thought to mainly involve its metabolic activation, forming 1,2-naphthoquinone (1,2-NQ), which can modify proteins through chemical modifications. In the present study, we examined the effect of 1,2-NQ on aggregation of crystallins (cry) associated with cataract. Incubation of bovine β-cry with 1,2-NQ caused covalent modification of β-cry at Cys117 and Lys125 accompanied by reduction in its thiol content, resulting in a concentration- and temperature-dependent aggregation of β-cry, whereas only little aggregation of α-cry induced by 1,2-NQ was seen. Interestingly, addition of α-cry to the reaction mixture of β-cry and 1,2-NQ markedly blocked β-cry aggregation induced by 1,2-NQ in a concentration-dependent manner. These results suggest that β-cry predominantly undergoes chemical modification by 1,2-NQ, causing its aggregation, which is suppressed by the chaperone-like protein, α-cry. This β-cry aggregation may be, at least in part, involved in the induction of cataract caused by 1,2-NQ.

Small heat shock proteins (sHSPs) emerged early in evolution and occur in all domains of life and nearly in all species, including humans. Mutations in four sHSPs (HspB1, HspB3, HspB5, HspB8) are associated with neuromuscular disorders. The aim of this study is to investigate the evolutionary forces shaping these sHSPs during vertebrate evolution. We performed comparative evolutionary analyses on a set of orthologous sHSP sequences, based on the ratio of non-synonymous: synonymous substitution rates for each codon. We found that these sHSPs had been historically exposed to different degrees of purifying selection, decreasing in this order: HspB8 > HspB1, HspB5 > HspB3. Within each sHSP, regions with different degrees of purifying selection can be discerned, resulting in characteristic selective pressure profiles. The conserved α-crystallin domains were exposed to the most stringent purifying selection compared to the flanking regions, supporting a 'dimorphic pattern' of evolution. Thus, during vertebrate evolution the different sequence partitions were exposed to different and measurable degrees of selective pressures. Among the disease-associated mutations, most are missense mutations primarily in HspB1 and to a lesser extent in the other sHSPs. Our data provide an explanation for this disparate incidence. Contrary to the expectation, most missense mutations cause dominant disease phenotypes. Theoretical considerations support a connection between the historic exposure of these sHSP genes to a high degree of purifying selection and the unusual prevalence of genetic dominance of the associated disease phenotypes. Our study puts the genetics of inheritable sHSP-borne diseases into the context of vertebrate evolution.

22 páginas, 8 figuras, Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease., The Spanish Ministerio de Ciencia e Innovación (Projects PID2019-108782RB-C22 and PID2019-108782RB-C21), Spanish Ministerio de Economía, Industria y Competitividad (Projects AGL2015-67023-C2-2-R and AGL2015-67023-C2-1-R), and the Project “CLU-2019-05 – IRNASA/CSIC Unit of Excellence”, funded by the Consejería de Educación, Junta de Castilla y León and co-financed by the European Union (ERDF “Europe drives our growth”). the JIN project ‘ULYSSES’ (RTI2018-093463-J-100) funded by the Spanish Ministerio de Ciencia, Innovación y Universidades.

To clarify the effect of a previously identified single nucleotide polymorphism (SNP; rs76740365 GA) in the exon-3 of theThe human recombinant wild-type and mutantThe mass spectrometric detection revealed that E156K mutation had no significant effect on the apparent molecular mass of theTaken together, our results for the first time showed that E156K mutation in

Background: An extensive study of the folding and stability of proteins and their complexes has revealed a number of problems and questions that need to be answered. One of them is the effect of chaperones on the process of fibrillation of various proteins and peptides. Methods: We studied the effect of molecular chaperones, such as GroEL and α-crystallin, on the fibrillogenesis of the Aβ(1-42) peptide using electron microscopy and surface plasmon resonance. Results: Recombinant GroEL and Aβ(1-42) were isolated and purified. It was shown that the assembly of GroEL occurs without the addition of magnesium and potassium ions, as is commonly believed. According to the electron microscopy results, GroEL insignificantly affects the fibrillogenesis of the Aβ(1-42) peptide, while α-crystallin prevents the elongation of the Aβ(1-42) peptide fibrils. We have demonstrated that GroEL interacts nonspecifically with Aβ(1-42), while α-crystallin does not interact with Aβ(1-42) at all using surface plasmon resonance. Conclusion: The data obtained will help us understand the process of amyloid formation and the effect of various components on it.

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3–6, 9–12 and 15–18 months after baseline) were performed in 41 patients who were classified empirically as ‘high’ (HR-T/HR-B) or ‘low’ (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA.

Acetylation of lysine residues occurs in lens proteins. Previous studies have shown an improvement in the chaperone activity of αA-crystallin upon acetylation. Sirtuins are NAD(+)-dependent enzymes that can deacylate proteins. The roles of sirtuins in regulating the acetylation of lens proteins and their impacts on the function of α-crystallin are not known. Here, we detected sirtuin activity in mouse lenses, and SIRT3 and SIRT5 were present primarily in the mitochondria of cultured primary mouse lens epithelial cells. Western blotting showed higher levels of protein acetylation in the lenses of SIRT3 KO and SIRT5 KO mice than in lenses of WT mice. Mass spectrometry analyses revealed a greater number of acetylated lysine residues in α-crystallin isolated from the SIRT3 and SIRT5 KO lenses than from WT lenses. α-Crystallin isolated from SIRT3 and SIRT5 KO lenses displayed a higher surface hydrophobicity and higher chaperone activity than the protein isolated from WT lenses. Thus, SIRTs regulate the acetylation levels of crystallins in mouse lenses, and acetylation in lenses enhances the chaperone activity of α-crystallin.

One‐third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single‐center prospective observational study, we analyzed IgG‐antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA‐binding protein 1 (MDP1) and alpha‐crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18‐month follow‐up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.

Several studies reported that α-crystallin concentrations in the eye lens cytoplasm decrease with a corresponding increase in membrane-bound α-crystallin with age and cataracts. The influence of the lipid and cholesterol composition difference between cortical membrane (CM) and nuclear membrane (NM) on α-crystallin binding to membranes is still unclear. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the α-crystallin binding to bovine CM and NM derived from the total lipids extracted from a single lens. Compared to CMs, NMs have a higher percentage of membrane surface occupied by α-crystallin and binding affinity, correlating with less mobility and more order below and on the surface of NMs. α-Crystallin binding to CM and NM decreases mobility with no significant change in order and hydrophobicity below and on the surface of membranes. Our results suggest that α-crystallin mainly binds on the surface of bovine CM and NM and such surface binding of α-crystallin to membranes in clear and young lenses may play a beneficial role in membrane stability. However, with decreased cholesterol content within the CM, which mimics the decreased cholesterol content in the cataractous lens membrane, α-crystallin binding increases the hydrophobicity below the membrane surface, indicating that α-crystallin binding forms a hydrophobic barrier for the passage of polar molecules, supporting the barrier hypothesis in developing cataracts.

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged β- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.

Spodoptera frugiperda (J. E. Smith) is a highly adaptable polyphagous migratory pest in tropical and subtropical regions. Small heat shock proteins (sHsps) are molecular chaperones that play important roles in the adaptation to various environment stressors. The present study aimed to clarify the response mechanisms of S. frugiperda to various environmental stressors. We obtained five S. furcifera sHsp genes (SfsHsp21.3, SfsHsp20, SfsHsp20.1, SfsHsp19.3, and SfsHsp29) via cloning. The putative proteins encoded by these genes contained a typical α-crystallin domain. The expression patterns of these genes during different developmental stages, in various tissues of male and female adults, as well as in response to extreme temperatures and UV-A stress were studied via real-time quantitative polymerase chain reaction. The results showed that the expression levels of all five SfsHsp genes differed among the developmental stages as well as among the different tissues of male and female adults. The expression levels of most SfsHsp genes under extreme temperatures and UV-A-induced stress were significantly upregulated in both male and female adults. In contrast, those of SfsHsp20.1 and SfsHsp19.3 were significantly downregulated under cold stress in male adults. Therefore, the different SfsHsp genes of S. frugiperda play unique regulatory roles during development as well as in response to various environmental stressors.

It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (K(a)) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the K(a) of α-crystallin binding to individual PL membranes followed the trends: K(a)(PC) > K(a)(SM) > K(a)(PS) > K(a)(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the K(a) of α-crystallin binding to two-component mixtures of PL membranes followed the trends: K(a)(SM/PE) > K(a)(SM/PS) > K(a)(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup’s size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes.

New evidence has been emerging that antibodies can be protective in various experimental models of tuberculosis. Here, we report on protection against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection using a combination of the human monoclonal IgA 2E9 antibody against the alpha-crystallin (Acr, HspX) antigen and mouse interferon-gamma in mice transgenic for the human IgA receptor, CD89. The effect of the combined mucosal IgA and IFN-γ; treatment was strongest (50-fold reduction) when therapy was applied at the time of infection, but a statistically significant reduction of lung bacterial load was observed even when the therapy was initiated once the infection had already been established. The protection involving enhanced phagocytosis and then neutrophil mediated killing of infected cells was IgA isotype mediated, because treatment with an IgG version of 2E9 antibody was not effective in human IgG receptor CD64 transgenic mice. The Acr antigen specificity of IgA antibodies for protection in humans has been indicated by their elevated serum levels in latent tuberculosis unlike the lack of IgA antibodies against the virulence-associated MPT64 antigen. Our results represent the first evidence for potential translation of mucosal immunotherapy for the management of MDR-TB.

Cataract is one of the major causes of blindness worldwide. Several factors including post-translational modification, thermal and solar radiations promote cataractogenesis. The camel lens proteins survive very harsh desert conditions and resist cataractogenesis. The folding and aggregation mechanism of camel lens proteins are poorly characterized. The camel lens contains three ubiquitous crystallins (α-, β-, and γ-crystallin) and a novel protein (ζ-crystallin) in large amounts. In this study, a sequence similarity search of camel α-crystallin with that of other organisms showed that the camel αB-crystallin consists of an extended N-terminal domain. Our results indicate that camel α-crystallin efficiently prevented aggregation of ζ-crystallin, with or without an obligate cofactor up to 89 °C. It performed a quick and efficient holdase function irrespective of the unfolding stage or aggregation. Camel α-crystallin exhibits approximately 20% chaperone activity between 30 and 40 °C and is completely activated above 40 °C. Camel α-crystallin underwent a single reversible thermal transition without loss of β-sheet secondary structure. Intrinsic tryptophan fluorescence and ANS binding experiments revealed two transitions which corresponded to activation of its chaperone function. In contrast to earlier studies, camel α-crystallin completely protected lens proteins during thermal stress.

Astrocytes are key players in CNS neuroinflammation and neuroregeneration that may help or hinder recovery, depending on the context of the injury. Although pro-inflammatory factors that promote astrocyte-mediated neurotoxicity have been shown to be secreted by reactive microglia, anti-inflammatory factors that suppress astrocyte activation are not well-characterized. Olfactory ensheathing cells (OECs), glial cells that wrap axons of olfactory sensory neurons, have been shown to moderate astrocyte reactivity, creating an environment conducive to regeneration. Similarly, astrocytes cultured in medium conditioned by cultured OECs (OEC-CM) show reduced nuclear translocation of nuclear factor kappa-B (NFκB), a pro-inflammatory protein that induces neurotoxic reactivity in astrocytes. In this study, we screened primary and immortalized OEC lines to identify these factors and discovered that Alpha B-crystallin (CryAB), an anti-inflammatory protein, is secreted by OECs via exosomes, coordinating an intercellular immune response. Our results showed that: (a) OEC exosomes block nuclear NFκB translocation in astrocytes while exosomes from CryAB-null OECs could not; (b) OEC exosomes could be taken up by astrocytes, and (c) CryAB treatment suppressed neurotoxicity-associated astrocyte transcripts. Our results indicate CryAB, as well as other factors secreted by OECs, are potential agents that can ameliorate, or even reverse, the growth-inhibitory environment created by neurotoxic reactive astrocytes following CNS injuries.

One of the most crowded biological environments is the eye lens which contains a high concentration of crystallin proteins. The molecular chaperones αB-crystallin (αBc) with its lens partner αA-crystallin (αAc) prevent deleterious crystallin aggregation and cataract formation. However, some forms of cataract are associated with structural alteration and dysfunction of αBc. While many studies have investigated the structure and function of αBc under dilute in vitro conditions, the effect of crowding on these aspects is not well understood despite its in vivo relevance. The structure and chaperone ability of αBc under conditions that mimic the crowded lens environment were investigated using the polysaccharide Ficoll 400 and bovine γ-crystallin as crowding agents and a variety of biophysical methods, principally contrast variation small-angle neutron scattering. Under crowding conditions, αBc unfolds, increases its size/oligomeric state, decreases its thermal stability and chaperone ability, and forms kinetically distinct amorphous and fibrillar aggregates. However, the presence of αAc stabilizes αBc against aggregation. These observations provide a rationale, at the molecular level, for the aggregation of αBc in the crowded lens, a process that exhibits structural and functional similarities to the aggregation of cataract-associated αBc mutants R120G and D109A under dilute conditions. Strategies that maintain or restore αBc stability, as αAc does, may provide therapeutic avenues for the treatment of cataract.

Cataracts are a major cause of blindness worldwide and commonly occur in individuals over 70 years old. Cataracts can also appear earlier in life due to genetic mutations. The lens proteins, αA- and αB-crystallins, are chaperone proteins that have important roles maintaining protein solubility to prevent cataract formation. Mutations in the CRYAA and CRYAB crystallin genes are associated with autosomal dominant early onset human cataracts. Although studies about the proteomic and genomic changes that occur in cataracts have been reported, metabolomics studies are very limited. Here, we directly investigated cataract metabolism using gas-chromatography-mass spectrometry (GC-MS) to analyze the metabolites in adult Cryaa-R49C and Cryab-R120G knock-in mouse lenses. The most abundant metabolites were myo-inositol, L-(+)-lactic acid, cholesterol, phosphate, glycerol phosphate, palmitic and 9-octadecenoic acids, α-D-mannopyranose, and β-D-glucopyranose. Cryaa-R49C knock-in mouse lenses had a significant decrease in the number of sugars and minor sterols, which occurred in concert with an increase in lactic acid. Cholesterol composition was unchanged. In contrast, Cryab-R120G knock-in lenses exhibited increased total amino acid content including valine, alanine, serine, leucine, isoleucine, glycine, and aspartic acid. Minor sterols, including cholest-7-en-3-ol and glycerol phosphate were decreased. These studies indicate that lenses from Cryaa-R49C and Cryab-R120G knock-in mice, which are models for human cataracts, have unique amino acid and metabolite profiles.

Small heat shock proteins (shsps) are conserved across invertebrate species. They are implicated in the modulation of various biological processes, such as immune responses, abiotic stress tolerance metamorphosis, and embryonic development. Herein, we identified a heat shock protein 20 from the red swamp crayfish, Procambarus clarkii (named as Pc-Hsp20), and performed in vivo studies to elucidate its physiological functions in the innate immunity. The open reading frame of Pc-Hsp20 was 609 base pair, encoding a protein of 202 amino acid residues with a hsp20/alpha crystallin family domain. Pc-Hsp20 was ubiquitously expressed in various tissues; however, it was highest in the hepatopancreas. The challenge with immune elicitors remarkably enhanced the transcript level of Pc-Hsp20 in the hepatopancreas when compared with the control. Administration of double-stranded RNA could significantly reduce expression of the Pc-Hsp20 mRNAs, and most of the immune-related genes expression enhanced with a variable concentration in the hepatopancreas. Altogether, these results suggest that Pc-Hsp20 may participate in innate immunity against microbial pathogens.

Potent neuroprotective effects of photobiomodulation with 670 nm red light (RL) have been demonstrated in several models of retinal disease. RL improves mitochondrial metabolism, reduces retinal inflammation and oxidative cell stress, showing its ability to enhance visual function. However, the current knowledge is limited to the main hypothesis that the respiratory chain complex IV, cytochrome c oxidase, serves as the primary target of RL. Here, we demonstrate a comprehensive cellular, molecular, and functional characterization of neuroprotective effects of 670 nm RL and 810 nm near-infrared light (NIRL) on blue light damaged murine primary photoreceptors. We show that respiratory chain complexes I and II are additional PBM targets, besides complex IV, leading to enhanced mitochondrial energy metabolism. Accordingly, our study identified mitochondria related RL- and NIRL-triggered defense mechanisms promoting photoreceptor neuroprotection. The observed improvement of mitochondrial and extramitochondrial respiration in both inner and outer segments is linked with reduced oxidative stress including its cellular consequences and reduced mitochondria-induced apoptosis. Analysis of regulatory mechanisms using gene expression analysis identified upregulation &alpha, crystallins that indicate enhanced production of proteins with protective functions that point to the rescued mitochondrial function. The results support the hypothesis that energy metabolism is a major target for retinal light therapy.