Massive increase in the stiffness of the human lens nucleus with age: the basis for presbyopia?

Purpose: To determine the stiffness of different regions of human lenses as a function of age, and to correlate the biophysical measurements in the lens center with nuclear water content. Methods: A custom made probe fitted to a dynamic mechanical analyzer was employed to measure stiffness values at 1 mm increments across equatorial sections of individual human lenses. Thermogravimetric analysis was used to determine the percentage water content in the nuclei of human lenses. Results: There was a pronounced increase in lens stiffness over the age range from 14 to 78. In the nucleus, stiffness values varied almost 1,000 fold over this age range, with the largest change observed in lenses between the ages of 20 to 60. Nuclear stiffness values increased on average by a factor of 450. By contrast, in the cortex the average increase in stiffness was approximately 20 fold over this same time period. In lenses younger than age 30, the nucleus was found to be softer than the cortex. This was true for all six lenses examined. In contrast all lenses older than 30 were characterized by having nuclear values higher than those of the cortex. In lenses over the age of 50, the lens nucleus was typically an order of magnitude more rigid than that of the cortex. The crossover age, when the cortical and nuclear stiffness values were similar, was in the 30s. There was no significant change in the water content of the human lens nucleus from age 13 to age 82. Conclusions: There is a marked increase in the stiffness of the human lens with age. This is most pronounced in the nucleus. Since in vivo data indicate that the nucleus must change shape significantly during accommodation, it is highly likely that these measured changes in physical properties will markedly diminish the ability of the lens to accommodate, and thus may be a major contributing factor to presbyopia. Since there was no measurable difference in the water contents of the nuclear regions of the lenses, this marked increase in stiffness is not due to compaction of the lens nucleus

Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins

Conspectus: Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer\u27s, Parkinson\u27s, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland

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. Fibre 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 N-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 transport mechanism of the mitochondrial ADP/ATP carrier

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou

Can The Fact That Myelin Proteins Are Old And Break Down Explain The Origin Of Multiple Sclerosis In Some People?

Recent discoveries may change the way that multiple sclerosis (MS) is viewed, particularly with regard to the reasons for the untoward immune response. The fact that myelin proteins are long-lived, and that by the time we are adults, they are extensively degraded, alters our perspective on the reasons for the onset of autoimmunity and the origin of MS. For example, myelin basic protein (MBP) from every human brain past the age of 20 years, is so greatly modified, that it is effectively a different protein from the one that was laid down in childhood. Since only a subset of people with such degraded MBP develop MS, a focus on understanding the mechanism of immune responses to central nervous system (CNS) antigens and cerebral immune tolerance appear to be worthwhile avenues to explore. In accord with this, it will be productive to examine why all people, whose brains contain large quantities of a foreign antigen , do not develop MS. Importantly for the potential causation of MS, MBP from MS patients breaks down differently from the MBP in aged controls. If the novel structures formed in these MS-specific regions are particularly antigenic, it could help explain the origin of MS. If verified, these findings could provide an avenue for the rational synthesis of drugs to prevent and treat MS

Molecular Processes Implicated in Human Age-Related Nuclear Cataract

Human age-related nuclear cataract is commonly characterized by four biochemical features that involve modifications to the structural proteins that constitute the bulk of the lens: coloration, oxidation, insolubility, and covalent cross-linking. Each of these is progressive and increases as the cataract worsens. Significant progress has been made in understanding the origin of the factors that underpin the loss of lens transparency. Of these four hallmarks of cataract, it is protein-protein cross-linking that has been the most intransigent, and it is only recently, with the advent of proteomic methodology, that mechanisms are being elucidated. A diverse range of cross-linking processes involving several amino acids have been uncovered. Although other hypotheses for the etiology of cataract have been advanced, it is likely that spontaneous decomposition of the structural proteins of the lens, which do not turn over, is responsible for the age-related changes to the properties of the lens and, ultimately, for cataract. Cataract may represent the first and best characterized of a number of human age-related diseases where spontaneous protein modification leads to ongoing deterioration and, ultimately, a loss of tissue function

Protein-Bound UV Filters in Normal Human Lenses: The Concentration of Bound UV Filters Equals That of Free UV Filters in the Center of Older Lenses

PURPOSE. To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. METHODS. Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. RESULTS. The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total proteinbound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. CONCLUSIONS. The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people. (Invest Ophthalmol Vis Sci. 2007;48:1718 -1723 DOI: 10.1167DOI: 10. /iovs.06-1134 T he primate lens is unique among both other primate tissues and the lenses of other species, in that it synthesizes 3-hydroxykynurenine glucoside (3OHKG) as a UV filter from the amino acid tryptophan (Trp). 1 The immediate precursors kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) are metabolites found in all organs of the body. All three of these Trp metabolites are unstable at physiological pH and undergo side chain deamination to yield ␣,␤-unsaturated ketones that are prone to nucleophilic attack. 2 In tissues that contain sufficient glutathione (GSH), it is likely that GSH will react with the deamination products before they bind to proteins 3 and that the adducts thus formed will diffuse out of the lens. In the interior of the lens, once the barrier to diffusion forms at middle age, 4 -6 the nucleus becomes a partially uncoupled region in which metabolites spend a longer time than in the young lens. This factor leads to an environment in the older lens nucleus that favors greater decomposition of intrinsically unstable molecules. Coupled with a diminished flux of GSH from the cortex, this results in increased covalent binding of UV filters to nuclear proteins after middle age. In this study, we set out to examine the levels of all three bound UV filters in normal lenses using a novel assay system in which lens proteins were incubated with excess GSH at pH 9.5. 9 Under these conditions, the UV filters that are attached to proteins are released, and the GSH adducts thus formed can be quantified by HPLC. UV filters were found to be covalently bound to proteins from all lenses older than 50 years. Such posttranslational modifications may have important consequences in terms of the susceptibility of the proteins to photooxidation and, in the case of 3OHKyn, the sensitivity to an oxidative environment such as that in the nuclei of lenses with age-related nuclear cataract. METHODS Purified water (purified to 18.2 M⍀/cm 2 ; Milli-Q; Millipore, Bedford, MA) was used in the preparation of all solutions. All organic solvents were HPLC grade (Ajax, Auburn, NSW, Australia). 3OHKyn, reduced GSH, trifluoroacetic acid (TFA), and guanidine HCl were obtained from Sigma-Aldrich (St. Louis, MO). Reversed-Phase HPLC Reversed-phase (RP)-HPLC was performed on a Shimadzu system (Kyoto, Japan). For analytical scale separations, a column (Jupiter, 5 m, C18, 300 Å, 250 ϫ 4.6 mm; Phenomenex, Torrance, CA) was used with the following mobile phase conditions: solvent A (aqueous 0.1% vol/vol TFA) for 5 minutes followed by a linear gradient of 0% to 50% solvent B (80% vol/vol acetonitrile/H 2 O, 0.1% vol/vol TFA) over 20 minutes, followed by a linear gradient of 50% to 100% B over 15 minutes and re-equilibration in the aqueous phase for 15 minutes. The flow rate was 0.5 mL/min and with detection at 360 nm. From th

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

Old proteins are widely distributed in the body. Over time, they deteriorate and many spontaneous reactions, for example isomerisation of Asp and Asn, can be replicated by incubation of peptides under physiological conditions. One of the signatures of long-lived proteins that has proven to be difficult to replicate in vitro is cleavage on the N-terminal side of Ser residues, and this is important since cleavage at Ser, and also Thr, has been observed in a number of human proteins. In this study, the autolysis of Ser- and Thr-containing peptides was investigated with particular reference to discovering factors that promote cleavage adjacent to Ser/Thr at neutral pH. It was found that zinc catalyses cleavage of the peptide bond on the N-terminal side of Ser residues and further that this process is markedly accelerated if a His residue is adjacent to the Ser. NMR analysis indicated that the imidazole group co-ordinates zinc and that once zinc is co-ordinated, it can polarize the carbonyl group of the peptide bond in a manner analogous to that observed in the active site of the metalloexopeptidase, carboxypeptidase A. The hydroxyl side chain of Ser/Thr is then able to cleave the adjacent peptide bond. These observations enable an understanding of the origin of common truncations observed in long-lived proteins, for example truncation on the N-terminal side of Ser 8 in Abeta, Ser 19 in alpha B crystallin and Ser 66 in alpha A crystallin. The presence of zinc may therefore significantly affect the long-term stability of cellular proteins

Large-scale binding of a-crystallin to cell membranes of aged normal human lenses: a phenomenon that can be induced by mild thermal stress

PURPOSE. With age, large amounts of crystallins become associated with fiber cell membranes in the human lens nucleus, and it has been proposed that this binding of protein may lead to the obstruction of membrane pores and the onset of a barrier to diffusion. This study focused on membrane binding within the barrier region and the outermost lens cortex. METHODS. Human lenses across the age range were used, and the interaction of crystallins with membranes was examined using sucrose density gradient centrifugation, two-dimensional gel electrophoresis, and amine-reactive isobaric tagging technology. Lipids were quantified using shotgun lipidemics. RESULTS. Binding of proteins to cell membranes in the barrier region was found to be different from that in the lens nucleus because in the barrier and outer cortical regions, only one high-density band formed. Most of the membrane-associated protein in this high-density band was alpha-crystallin. Mild thermal stress of intact young lenses led to pronounced membrane binding of proteins and yielded a sucrose density pattern in all lens regions that appeared to be identical with that from older lenses. CONCLUSIONS. alpha-Crystallin is the major protein that binds to cell membranes in the barrier region of lenses after middle age. Exposure of young human lenses to mild thermal stress results in large-scale binding of alpha-crystallin to cell membranes. The density gradient profiles of such heated lenses appear to be indistinguishable from those of older normal lenses. The data support the hypothesis that temperature may be a factor responsible for age-related changes to the human lens. (Invest Ophthalmol Vis Sci. 2010;51:5145-5152) DOI:10.1167/iovs.10-526