Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry.

Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel reaction monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type mice and mice with deafness mutations affecting hair-bundle proteins.

Superior prevention of calcium ionophore cataract by E64d.

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.

Calpain II induced insolubilization of lens beta-crystallin polypeptides may induce cataract.

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca2+, it was limited to 6% of the soluble protein present and resulted from precipitation of proteolyzed beta-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble beta-crystallin polypeptides produced by calpain II were similar to insoluble beta-crystallin polypeptides found in cataractous lenses. Trypsin also caused insolubilization of beta-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from beta-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.

Lysine acetyltransfer supports platelet function.

BACKGROUND AND OBJECTIVES: The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function. METHODS AND RESULTS: Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of the identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments showed that platelets express p300, which is activated in an Src kinase-dependent manner upon platelet stimulation with the platelet glycoprotein VI agonist collagen-related peptide (CRP). Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin, and cortactin, as well as F-actin polymerization, integrin activation, and platelet aggregation. Super-resolution visualization of platelet actin-rich adhesion structures revealed abundant acK protein colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodium formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiologic role for lysine acetyltransferase activity in platelet function. CONCLUSION: Together, our findings reveal lysine acetyltransfer to be a potential regulator of platelet actin dynamics, and potential roles for lysine acetylation in the molecular coordination of platelet activation and function.

Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-ß and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Beta-crystallins insolubilized by calpain II in vitro contain cleavage sites similar to beta-crystallins insolubilized during cataract.

Incubation of soluble proteins from rat lens with the protease calpain II caused the precipitation of beta-crystallin polypeptides. Two-dimensional electrophoresis and sequence analysis identified beta-crystallin polypeptides both before and after their precipitation by calpain II. beta-crystallin polypeptides precipitated by calpain were cleaved at their NH2-terminal extensions. These cleavage sites were similar to cleavage sites occurring in beta-crystallin polypeptides precipitated during formation of experimental cataract induced by an overdose of selenite. These data suggested that calpain II caused beta-crystallin insolubilization during cataract formation, and indicated that the process can be mimicked in vitro.

Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis.

A single injection of 20 mumol sodium selenite/kg body weight in 10-day-old rats caused severe nuclear cataract within 4 days. By 4 days postselenite injection, nuclear calcium levels increased from 0.4 to 6.8 mmol/kg lens dry weight. The purpose of these experiments was to determine if this calcium increase was associated with proteolysis specifically in the lens nuclear region. Sodium dodecyl sulfate polyacrylamide electrophoresis of lens nuclear proteins following selenite injection showed: loss of 30, 27, and 26 K molecular weight polypeptides in the soluble fraction, loss of 83, 52, 30, 27, and 26 K polypeptides in the insoluble fraction, and loss of the major 26 K membrane protein. Gel chromatography of nuclear soluble proteins indicated a decrease in beta H and beta L crystallins following selenite injection. Two-hour in vitro incubation of nuclear lens homogenates with calcium duplicated many of the proteolytic changes occurring in lenses in vivo following selenite injection. Calcium induced proteolysis in vitro was inhibited by EGTA, leupeptin, and iodoacetate but was not inhibited by phenylmethylsulfonyl fluoride. These properties are similar to calcium activated protease (CAP) from other tissues. Activation of CAP, and subsequent degradation of nuclear proteins, may be causes of selenite cataract.

Cataract and the acceleration of calpain-induced beta-crystallin insolubilization occurring during normal maturation of rat lens.

PURPOSE: To determine if limited proteolysis of beta-crystallins is associated with insolubilization of proteins in rats lens during maturation and to test if the protease, calpain II, is involved. METHODS: Soluble and insoluble lens proteins from 4-day-old to 4-month-old rat lens cortexes and nuclei were separated by two-dimensional electrophoresis. The insoluble proteins from 4-month-old nuclei were electroblotted and the NH2 termini of proteins sequenced. Cleavage sites appearing at 4 months of age were compared to cleavage sites produced by purified calpain II and to cleavage sites appearing in cataracts induced by selenite in vivo or in lenses cultured with calcium ionophore A23187 or diamide. RESULTS: In solubilization of more than 50% of proteins occurred in the nucleus of the transparent rat lens by 4 months of age. The insoluble protein that formed contained an abundance of partially degraded beta-crystallin polypeptides missing portions of their NH2 terminal extensions. In contrast, these truncated beta-crystallins were largely absent from both the cortex and soluble fraction of the nucleus. The cleavage sites in the insoluble beta-crystallins appearing during maturation in the lens nucleus were similar to cleavage sites produced by purified calpain II and also similar to cleavage sites appearing in the insoluble protein of cataractous lenses. CONCLUSIONS: These results suggest that proteolysis of beta-crystallins by the protease calpain II contributes to protein insolubilization during lens maturation and that acceleration of this insolubilization process is associated with cataract formation in rodent lenses.

Crystallin degradation and insolubilization in regions of young rat lens with calcium ionophore cataract.

PURPOSE: To determine if the susceptibility of rat lenses to cataract formation in culture changes with increasing age and to investigate the regional differences in crystallin degradation and insolubilization during the formation of cataracts in cultured lenses. METHODS: Lenses from 4-week-old (young group) and 12-week-old (adult group) rats were divided into four subgroups: noncultured control, cultured control, cultured in calcium ionophore A23187, and cultured in ionophore plus calpain inhibitor E64. Lenses were cultured for 7 days, and the cortex and nucleus were homogenized and separated into water-soluble and water-insoluble fractions. Two-dimensional electrophoresis and N-terminal sequencing were then performed. RESULTS: Young lenses treated with ionophore produced thin cortical and dense nuclear opacities. Adult lenses treated with ionophore also developed thin cortical opacity, but no nuclear opacity was observed, even though a large increase in the concentration of insoluble protein occurred. Two-dimensional electrophoresis and sequencing suggested that calpain caused protein degradation in the cortex region. However, unlike nuclear opacity, the formation of opacity in the cortex was not inhibited by E64 in young or adult lenses. CONCLUSIONS: Calpain was activated, and crystallins were proteolyzed in the cortex of ionophore-treated lenses. However, cortical opacity was not the result of proteolysis by calpain. Maturation also decreased the susceptibility of rat lens nucleus to calcium ionophore cataract.

Amelioration of cataracts and proteolysis in cultured lenses by cysteine protease inhibitor E64.

Cataracts were produced in cultured rat lenses by either 10 microM calcium ionophore A23187, 25 microM sodium selenite, or 30 mM xylose. E64, an inhibitor of cysteine proteases, such as calpain (EC, 3.4.22.17), reduced severity of cataract and proteolysis of crystallins when included at a 500 microM concentration in the culture medium along with cataractogenic agents. Calpain II enzyme activity and the amount of calpain antigen were decreased in the cytosol of cataractous lens. However, E64 caused an increase in the amount of an 80-kD calpain subunit associated with the ethyleneglycol-bis-(beta-aminoethylether) tetraacetic acid/ethylenediaminetetraacetic acid-washed insoluble proteins when lenses were incubated with cataractous agents. These data indicate that E64 was at least partially effective in inhibiting lens calpain, and that activation of lens calpain may involve binding to the insoluble fraction. These results provide strong evidence for the activation of calpain in rodent cataracts and suggest testing inhibitors of calpain as anticataract drugs.

Origin of urea-soluble protein in the selenite cataract. Role of beta-crystallin proteolysis and calpain II.

Nuclear cataract resulting from an overdose of selenite was characterized by a five-fold increase in nuclear urea-soluble protein. The origin of this urea-soluble protein was examined by two-dimensional electrophoresis, immunoblotting with monospecific antisera against rat lens crystallins, and tryptic mapping. Cataractous urea-soluble protein was primarily composed of insolubilized beta- and gamma-crystallin polypeptides. Polypeptides from cataractous urea-soluble protein, and normal beta L-crystallin aggregates were compared by tryptic mapping. Approximately 19% of the urea-soluble protein from opaque nuclei was composed of 24.7 and 24.0 K polypeptides derived by limited proteolysis of 26.5 K beta L-crystallin polypeptide. Incubation of 26.5 K beta-crystallin polypeptide with purified rat lens calpain II in vitro caused production of fragments with similar molecular weights to polypeptides found in cataractous lenses. These results support the hypothesis that proteolysis may contribute to formation of urea-soluble protein in selenite cataract.

Regional distribution of free calcium in selenite cataract: relation to calpain II.

The purpose of this experiment was to assess the roles of free, intracellular calcium and calcium-dependent neutral protease (calpain II, EC.34.22.17) in selenite nuclear cataract. Free calcium ion concentrations within lens nuclear fibers during selenite cataractogenesis increased to 3 microM on day 2 post-injection (clear lens) and to 108 microM at day 4 (nuclear cataract). Calpain II is known to be activated in vitro by calcium levels above 50 microM. Calpain II activity was present in the lens nucleus at time periods preceding formation of selenite cataract. These data suggested that after selenite injection, calpain II was activated by elevated free calcium in the nucleus, and that calpain II-induced proteolysis of nuclear proteins was an important mechanism in selenite cataract. Calpain II levels were also observed to decrease in the nucleus during selenite cataractogenesis, probably due to autolysis. This was supported by the finding that incubation of purified lens calpain II with 100 microM calcium caused partial inactivation of the protease.

Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice.

PURPOSE: The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS: Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day-old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS: Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of alphaA-crystallin. CONCLUSIONS: Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

Calpain II in human lens.

The purposes of the current study were to: determine if human lenses contain calpain II (EC.34.22.17) activity, measure the effect of aging and anatomical location on lens calpain II activity, and determine if human lenses contain the endogenous calpain inhibitor calpastatin. Both enzymatic and immunologic assays indicated that human lenses contained calpain II activity. Calpain II activity was highest in the cortex of lenses from young donors, and lowest in the nucleus of aged lenses, where it was sometimes nondetectable. In some cases, calpain II activity persisted in the nucleus of lenses from donors greater than 70 years of age. Human lenses also contained endogenous calpain inhibitor (calpastatin) in excess over calpain enzymatic activity. Calpastatin activity did not decrease during aging. Although human lenses contained approximately 3% of the calpain activity found in rat lenses, calpain II may still be a major endopeptidase in human lenses. Demonstration of calpain II in human lenses suggested that calpain II could be involved in both lens maturation and cataract formation.

Calpain in rat cornea.

This study was conducted to provide a description of calpain proteolytic enzyme (EC 3.4.22.17) in normal rat cornea and to document immunohistochemical changes in calpain distribution during maturation. Corneal soluble proteins were fractionated by diethylaminoethyl chromatography on high-performance liquid chromatography. Fractions were analyzed for calpain by enzyme-linked immunosorbent assay, immunoblotting, and caseinolytic enzyme activity with fluorescein isothiocyanate-labeled casein. Calpain II from the soluble fraction of 2-week-old and 3-month-old rat corneas eluted at a similar NaCl concentration (220-240 mmol/l) as calpain II from other tissues, was inhibited by E64, contained an 80-kilodalton subunit in immunoblots, and was present at specific activity of 473 units per gram of protein in 3-month-old rats and 801 units per gram of protein in 2-week-old rats. Calpain antigen also was present in the ethylenediaminetetraacetic acid and EGTA washed insoluble fraction of cornea. Calpain was found (by immunohistochemical analysis) in all layers of the epithelium but not in the stroma. Enzyme-linked immunsorbent assay, immunoblots, and immunohistochemical analysis showed that calpain in the whole cornea did not change with corneal maturation. It was hypothesized that calpain in the cornea may be involved in the turnover of epithelial cells during normal maturation.

Cleavage of beta crystallins during maturation of bovine lens.

PURPOSE: (1) Identify major crystallin proteins in fetal and adult bovine lens, (2) examine the N-termini of beta-crystallins for truncation, and (3) determine if the protease m-calpain (EC 3.4.22.17) is responsible for the cleavage of bovine beta-crystallins during maturation. METHODS: Crystallins from fetal and adult bovine lenses were analyzed by one and two-dimensional electrophoresis and Edman sequencing of separated proteins and their tryptic fragments. Identical techniques were used to analyze crystallins following their incubation with purified m-calpain. RESULTS: The identities of the major crystallins and several additional crystallin species missing portions of their N-terminal extensions were identified in the fetal bovine lens. Besides the previously identified form of betaB1 missing 15 residues from its N-terminus, forms of betaA3 >missing 11 and 22 residues were identified. With aging, the betaA3 (-22) species became a major protein in the adult bovine lens, and minor forms of betaB2 and betaB3 missing 8 and 22 residues from their N-termini, respectively, appeared. Purified m-calpain cleaved within the N-terminal extensions of bovine beta-crystallins and removed: 12 or 15 residues from betaB1; 8 residues from betaB2; 5 or 10 residues from betaB3; and 11 or 17 residues from betaA3. CONCLUSIONS: Based on the cleavage sites in vitro, m-calpain may be partially responsible for cleavage of bovine betaB1, betaB2, and betaA3 during lens maturation. However, the preference of m-calpain to remove 12 residues from betaB1, and 11 and 17 residues from betaA3, suggested that the betaB1 (-15) and betaA3 (-22) species found in vivo were produced by a different protease. This unidentified protease may have a preference for the asparagine-proline-X-proline sequence found in the N-terminal extensions of betaB1 and betaA3.

Local microdomain structure in the terminal extensions of betaA3- and betaB2-crystallins.

PURPOSE: Although the crystal structures of the core domains of bovine betaB2-crystallin have been determined and those of other betagamma-crystallins modeled, the positions of the N- and C-termini are not resolvable by X-ray crystallography. Here we model the possible structural organization of the terminal arms of mouse betaA3- and betaB2-crystallins and test this model against the results of partial proteolysis. METHODS: The secondary structure of the terminal extensions was predicted by 3 different methods, one a nearest-neighbor method modified to use overlapping sequence tripeptides. Recombinant betaA3- and betaB2-crystallins were expressed using baculovirus vectors in S. frugiperda Sf9 cells. Crystallins were sequenced by the Edman degradation method. RESULTS: The N-terminal extension of betaB2-crystallin includes a series of hydrophilic residues from Q-11 to Q-9 which have high propensity of a helical conformation. The N-terminal arm of betaA3-crystallin is also predicted to have two helical segments, from Q-24 to E-20 and M-13 to A-12. Partial characterization of the baculovirus extract showed a thiol protease inhibited by leupeptin and E-64. As predicted by the model, recombinant betaB2-crystallin subjected to partial proteolysis was cleaved adjacent to the helical domain, while the N-terminal cleavage site in recombinant betaA3-crystallin was within 1 residue of an interhelical junction. Our model also predicts the products of partial proteolytic degradation of betaB2- and betaA3-crystallins from human, rat, bovine and chicken lenses incubated with the protease m-calpain. CONCLUSIONS: These results suggest the existence of local microdomain structures in the N- and C-terminal extensions of betaA3- and betaB2-crystallins, which appear to be more susceptible to proteolytic degradation in regions adjacent to these putative domains.

Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections.

Collagenase is a potential virulence factor shown to be expressed by Porphyromonas gingivalis associated with periodontal disease. The purpose of this study was to use the polymerase chain reaction (PCR) to detect the presence of the collagenase gene (prtC) in 21 strains of Porphyromonas species isolated from endodontic infections. Type strains for P. gingivalis (ATCC 33277), P. endodontalis (ATCC 35406), Prevotella intermedia (ATCC 25611), and Prevotella nigrescens (ATCC 33563) were used as controls. When PCR primers specific for the 16S ribosomal RNA gene of P. gingivalis or P. endodontalis were used, 16 of the strains were identified as P. gingivalis, and five strains were identified as P. endodontalis. The presence of the prtC gene for collagenase was detected using PCR. Amplicons were analyzed by agarose gel electrophoresis, with an 815 bp amplicon representing the presence of the collagenase gene. Type strain ATCC 33277 and all 16 clinical isolates of P. gingivalis produced the collagenase gene amplicon. Neither type strain ATCC 35406 nor the five strains from clinical isolates of P. endodontalis produced the collagenase gene amplicon. These results indicate that P. gingivalis from endodontic infections possesses the prtC gene. P. endodontalis does not seem to exhibit prtC. The virulence of P. gingivalis may be related to its production of collagenase.

Occurrence of Prevotella nigrescens and Prevotella intermedia in infections of endodontic origin.

The occurrence of Prevotella intermedia and Prevotella nigrescens in endodontic infections was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell protein to distinguish between the species. Previous studies have shown an association between black-pigmented bacteria (BPB) and endodontic infections and that Prevotella intermedia (previously known as Bacteroides intermedius) was the most commonly isolated BPB. Recently, however, strains identified as P. intermedia were shown to in fact be composed of two separate species, P. intermedia and P. nigrescens. Fifty-six strains of BPB isolated from endodontic infections and previously identified as P. intermedia were used in this study. Following SDS-PAGE, P. nigrescens showed a unique 18.6 kDa band that was used to differentiate P. nigrescens from P. intermedia. Of the 56 strains of BPB, 41 (73.2%) were identified as P. nigrescens and 15 (26.8%) as P. intermedia. This study confirms that P. nigrescens, and not P. intermedia, is the BPB most often isolated from infections of endodontic origin.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polymerase chain reaction for differentiation of Prevotella intermedia and Prevotella nigrescens.

Isolates previously thought to be Prevotella intermedia have been shown to be a closely related species now known as Prevotella nigrescens. The purpose of this study was to determine the efficacy of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) to differentiate endodontic isolates of P. nigrescens from P. intermedia. Fifty-six strains of black-pigmented bacteria isolated from endodontic infections and conventionally identified as P. intermedia were used in this study. Using SDS-PAGE, novel polypeptide bands were used to differentiate P. nigrescens from P. intermedia. PCR was accomplished with specific primers for the 16S ribosomal RNA gene of both strains. Of 56 endodontic isolates, 41 (73%) strains were identified by SDS-PAGE as P. nigrescens and 15 (27%) strains as P. intermedia. Of the 41 strains of P. nigrescens identified by SDS-PAGE, PCR identified 37 strains as P. nigrescens. Restriction endonuclease digestion of amplified 16S ribosomal RNA genes indicated that the remaining four strains originally identified by SDS-PAGE as P. nigrescens were actually strains of Prevotella distinct from P. nigrescens and P. intermedia. Of 15 strains of P. intermedia identified by SDS-PAGE, PCR identified 14 strains as P. intermedia; but, one strain was identified as P. nigrescens. The results indicated that PCR was a more precise method than SDS-PAGE to differentiate P. intermedia from P. nigrescens. This study confirms that P. nigrescens is more commonly isolated in pure culture from endodontic infections than P. intermedia.