Lanostane Triterpenoids with PTP1B Inhibitory and Glucose-Uptake Stimulatory Activities from Mushroom Fomitopsis pinicola Collected in North America.

A chemical investigation on the fruiting bodies of Fomitopsis pinicola led to the isolation and identification of 28 lanostane triterpenoids including 11 new compounds (1-11) and 17 known analogues (12-28). Their structures were elucidated by extensive one-dimensional NMR, two-dimensional NMR, and MS spectra. All isolates were tested for their anti-inflammatory activity, protein tyrosine phosphatase 1B (PTP1B) inhibitory activity in vitro, and effect on glucose uptake in insulin-resistant HepG2 cells. Compounds 1, 4, 22, 23, and 27 inhibited the nitric oxide released from the LPS-induced RAW 264.7 cell assay with IC50 values in the range of 21.4-27.2 µM. Compounds 18, 22, 23, and 28 showed strong PTP1B inhibitory activity with IC50 values in the range of 20.5-29.9 µM, comparable to that of the positive control of oleanolic acid (15.0 µM). Compounds 18 and 22 were confirmed to be good competitive inhibitors of PTP1B by kinetic analysis. In addition, compounds 18, 22, and 28 were found to stimulate glucose uptake in the insulin-resistant HepG2 cells in the dose from 6.25 to 100 µM. These findings indicated the potential of F. pinicola in the development of functional food or medicine for the prevention and treatment of diabetes.

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

A mathematical-descriptor of tumor-mesoscopic-structure from computed-tomography images annotates prognostic- and molecular-phenotypes of epithelial ovarian cancer.

The five-year survival rate of epithelial ovarian cancer (EOC) is approximately 35-40% despite maximal treatment efforts, highlighting a need for stratification biomarkers for personalized treatment. Here we extract 657 quantitative mathematical descriptors from the preoperative CT images of 364 EOC patients at their initial presentation. Using machine learning, we derive a non-invasive summary-statistic of the primary ovarian tumor based on 4 descriptors, which we name "Radiomic Prognostic Vector" (RPV). RPV reliably identifies the 5% of patients with median overall survival less than 2 years, significantly improves established prognostic methods, and is validated in two independent, multi-center cohorts. Furthermore, genetic, transcriptomic and proteomic analysis from two independent datasets elucidate that stromal phenotype and DNA damage response pathways are activated in RPV-stratified tumors. RPV and its associated analysis platform could be exploited to guide personalized therapy of EOC and is potentially transferrable to other cancer types.

Baeyer-Villiger Monooxygenase-Mediated Synthesis of Esomeprazole As an Alternative for Kagan Sulfoxidation.

A wild-type Baeyer-Villiger monooxygenase was engineered to overcome numerous liabilities in order to mediate a commercial oxidation of pyrmetazole to esomeprazole, using air as the terminal oxidant in an almost exclusively aqueous reaction matrix. The developed enzyme and process compares favorably to the incumbent Kagan inspired chemocatalytic oxidation, as esomeprazole was isolated in 87% yield, in >99% purity, with an enantiomeric excess of >99%.

Origins of stereoselectivity in evolved ketoreductases.

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Fluoroscopy-based laser guidance system for linear surgical tool insertion depth control.

PURPOSE: In most orthopedic surgeries, knowing how far to insert surgical tools is crucial. The objective of this study was to provide guidance information on depth without tracking surgical tools. A previously developed laser guidance system for linear surgical tool insertion uses two laser beams that display the insertion point and orientation on the skin surface. However, the system only provides 4 degrees of freedom guidance (an entry point on the planned pathway line and the orientation) but do not inform surgeons on the ideal insertion depth. METHOD: A 5-DOF guidance method was developed to provide guidance information by direct projection onto the surgical area using laser beams without tracking markers. A position and orientation guidance performed by two laser beams and depth guidance performed by a single laser beam are appeared on the surgical area in turn. However, depth point appears on the surgical tool side face with some error because of tool radius. Using the actual depth position, insertion path vector and location of the laser sources, the correct depth point on the tool's surface is calculated by the proposed method. So, this system can indicate and navigate the 5-DOF which is planning path and the correct depth point. RESULTS: An evaluation of the accuracy of depth guidance revealed a depth guidance error of 0.55±0.29 mm and results from phantom target insertions revealed overall system accuracies of 1.44 ± 1.09 mm, 0.91° ± 0.82°. In addition, overall system accuracies of application feasibility experiment under the X-ray condition were 1.94 ± 0.98mm, 1.39° ± 1.30°. CONCLUSION: A new surgical tool depth insertion method was developed using a fluorolaser guidance system. This tool informs surgeons of the surgical tool tip depth assuming that the insertion point and orientation are correct. The new method was tested successfully in vitro.

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

A fluorolaser navigation system to guide linear surgical tool insertion.

PURPOSE: Conventional navigation systems for minimally invasive orthopedic surgery require a secondary monitor to display guidance information generated with CT or MRI images. Newer systems use augmented reality to project surgical plans into binocular glasses. These surgical procedures are often mentally challenging and cumbersome to perform. METHOD: A comprehensive surgical navigation system for direct guidance while minimizing radiation exposure was designed and built. System accuracy was evaluated using in vitro needle insertion experiments. The fluoroscopic-based navigation technique is combined with an existing laser guidance technique. As a result, the combined system is capable of surgical planning using two or more X-ray images rather than CT or MRI scans. Guidance information is directly projected onto the patient using two laser beams and not via a secondary monitor. RESULTS: We performed 15 in vitro needle insertion experiments as well as 6 phantom pedicle screw insertion experiments to validate navigation system accuracy. The planning accuracy of the system was found to be 2.32 mm and 2.28°, while its overall guidance accuracy was found to be 2.40 mm and 2.39°. System feasibility was demonstrated by successfully performing percutaneous pin insertion on phantoms. CONCLUSION: Quantitative and qualitative evaluations of the fluorolaser navigation system show that it can support accurate guidance and intuitive surgical tool insertion procedures without preoperative 3D image volumes and registration processes.

Average sound speed estimation using speckle analysis of medical ultrasound data.

PURPOSE: Most ultrasound imaging systems assume a pre-determined sound propagation speed for imaging. However, a mismatch between assumed and real sound speeds can lead to spatial shift and defocus of ultrasound image, which may limit the applicability of ultrasound imaging. The estimation of real sound speed is important for improving positioning accuracy and focus quality of ultrasound image. METHOD: A novel method using speckle analysis of ultrasound image is proposed for average sound speed estimation. Firstly, dynamic receive beam forming technology is employed to form ultrasound images. These ultrasound images are formed by same pre-beam formed radio frequency data but using different assumed sound speeds. Secondly, an improved speckle analysis method is proposed to evaluate focus quality of these ultrasound images. Thirdly, an iteration strategy is employed to locate the desired sound speed that corresponds to the best focus quality image. RESULTS: For quantitative evaluation, a group of ultrasound data with 20 different structure patterns is simulated. The comparison of estimated and simulated sound speeds shows speed estimation errors to be -0.7 ± 2.54 m/s and -1.30 ± 5.15 m/s for ultrasound data obtained by 128- and 64-active individual elements linear arrays, respectively. Furthermore, we validate our method via phantom experiments. The sound speed estimation error is -1.52 ± 8.81 m/s. CONCLUSION: Quantitative evaluation proves that proposed method can estimate average sound speed accurately using single transducer with single scan.

Efficient, chemoenzymatic process for manufacture of the Boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization.

The key structural feature in Boceprevir, Merck's new drug treatment for hepatitis C, is the bicyclic [3.1.0]proline moiety "P2". During the discovery and development stages, the P2 fragment was produced by a classical resolution approach. As the drug candidate advanced through clinical trials and approached regulatory approval and commercialization, Codexis and Schering-Plough (now Merck) jointly developed a chemoenzymatic asymmetric synthesis of P2 where the net reaction was an oxidative Strecker reaction. The key part of this reaction sequence is an enzymatic oxidative desymmetrization of the prochiral amine substrate.

Oligomerization with wt αA- and αB-crystallins reduces proteasome-mediated degradation of C-terminally truncated αA-crystallin.

PURPOSE: We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated αA-crystallin. The objective of this work was to determine the effects of wt αA- and αB-crystallins on the degradation of C-terminally truncated αA-crystallin (αA(1-162)) and vice versa. METHODS: Recombinant wt αA, αB, and αA(1-162) were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and coprecipitation assays. Protein substrates were labeled with (125)I and lens epithelial cell lysates were used as the source of the UPP for degradation assays. RESULTS: FRET, multiangle light scattering, and coprecipitation assays showed that αA(1-162) exchanged subunits with wt αA- or wt αB- crystallin to form hetero-oligomers. αA(1-162) was more susceptible than wt αA-crystallin to degradation by the UPP. When mixed with wt αA-crystallin at 1:1 or 1:4 (αA(1-162) : wt) ratios to form hetero-oligomers, the degradation of αA(1-162) was significantly decreased. Conversely, formation of hetero-oligomers with αA(1-162) enhanced the degradation of wt αA-crystallin. The presence of αA(1-162), but not wt αA-crystallin, decreased the degradation of wt αB-crystallin. CONCLUSIONS: αA(1-162) forms hetero-oligomers with wt αA- and αB-crystallins. Oligomerization with wt αA- or αB-crystallins reduces the susceptibility of αA(1-162) to degradation by the UPP. In addition, the presence of αA(1-162) in the hetero-oligomers also affects the degradation of wt αA- and αB-crystallins.

Wearable wrist activity monitor as an indicator of functional hand use in children with cerebral palsy.

AIM: New tools that capture hand function in everyday activities and contexts are needed for assessing children with hemiplegic cerebral palsy. This study evaluates a wearable wrist monitor and tests the hypothesis that wrist extension frequency (FreqE) is an appropriate indicator of functional hand use. METHOD: Fifteen children (four females, 11 males; age range 6-12y; mean age 10y [SD 2y]) with hemiplegia (seven at level I and eight at level II on the Manual Ability Classification System) participated in the Assisting Hand Assessment (AHA) while wearing the wrist monitor. FreqEs were captured via the wrist monitor and validated using video analysis. Correlations between FreqE and AHA scores were calculated and a multivariate linear regression was conducted to explore other measures of wrist activity. RESULTS: Wrist extensions observed in video analyses were reliably detected by the wrist monitor (intraclass correlation coefficient, r=0.88; p<0.001) and were strongly correlated with the AHA scores (r=0.93; p<0.001). AHA scores were significantly correlated with FreqE (r=0.80; p=0.001) and the range of wrist extensions/flexions (r=0.70; p=0.008). The multivariate linear regression combining the FreqE and range of wrist extensions/flexions yielded a strong correlation with AHA scores (r=0.84; p=0.0043). INTERPRETATION: The wearable wrist monitor may offer a convenient, valid alternative to observer reports for functional assessments of the hemiplegic hand in everyday contexts.

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins.

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components--ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)--suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, ßB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, ßB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.

Practical chiral alcohol manufacture using ketoreductases.

Over the past two years the application of ketoreductases in the commercial synthesis of chiral alcohols has undergone a revolution. Biocatalysts are now often the preferred catalyst for the synthesis of chiral alcohols via ketone reduction and are displacing reagents and chemocatalysts that only recently were considered break-through process solutions themselves. Tailor-made enzymes can now be generated from advanced, non-natural variants using HTP screening and modern molecular biology techniques. At the same time, global economic and environmental pressures direct industrial process development toward versatile platforms that can be applied to the different stages of product development. We will discuss the technologies that have emerged over the past years that have guided biocatalysis from the bottom of the toolbox, to the power tool of choice.

GeNMR: a web server for rapid NMR-based protein structure determination.

GeNMR (GEnerate NMR structures) is a web server for rapidly generating accurate 3D protein structures using sequence data, NOE-based distance restraints and/or NMR chemical shifts as input. GeNMR accepts distance restraints in XPLOR or CYANA format as well as chemical shift files in either SHIFTY or BMRB formats. The web server produces an ensemble of PDB coordinates for the protein within 15-25 min, depending on model complexity and completeness of experimental restraints. GeNMR uses a pipeline of several pre-existing programs and servers to calculate the actual protein structure. In particular, GeNMR combines genetic algorithms for structure optimization along with homology modeling, chemical shift threading, torsion angle and distance predictions from chemical shifts/NOEs as well as ROSETTA-based structure generation and simulated annealing with XPLOR-NIH to generate and/or refine protein coordinates. GeNMR greatly simplifies the task of protein structure determination as users do not have to install or become familiar with complex stand-alone programs or obscure format conversion utilities. Tests conducted on a sample of 90 proteins from the BioMagResBank indicate that GeNMR produces high-quality models for all protein queries, regardless of the type of NMR input data. GeNMR was developed to facilitate rapid, user-friendly structure determination of protein structures via NMR spectroscopy. GeNMR is accessible at http://www.genmr.ca.

Protein-protein interactions involving congenital cataract T5P gammaC-crystallin mutant: a confocal fluorescence microscopy study.

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects protein-protein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP-RFP (donor-acceptor) plasmid pairs was cotransfected into HeLa cells: gammaC-gammaC, gammaC-gammaCT5P, gammaCT5P-gammaCT5P, alphaA-gammaC, and alphaA-gammaCT5P. After culture, confocal fluorescence cell images were taken. Protein-protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P gammaC-crystallin contain many protein aggregates, but cells co-expressing with either gammaC- or alphaA-crystallin reduce the aggregation considerably. FRET determination indicates that gammaCT5P-gammaCT5P shows less protein-protein interaction than either gammaC-gammaC or gammaC-gammaCT5P. Cotransfection with alphaA-crystallin (alphaA-gammaC or alphaA-T5PgammaC) increases nFRET compared with gammaC-gammaC or gammaC-T5PgammaC. Our results demonstrate that T5P gammaC-crystallin shows more protein aggregates and less protein-protein interaction than WT gammaC-crystallin. Chaperone alphaA-crystallin can rescue T5P gammaC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein-protein interactions and increased aggregation from an insufficient amount of alpha-crystallin for protection.

Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.

PURPOSE: The R120G mutation of alphaB-crystallin is known to cause desmin-related myopathy, but the mechanisms underlying the formation of cataract are not clearly established. We hypothesize that alteration of protein-protein interaction between R120G alphaB-crystallin and lens intermediate filament proteins is one of the mechanisms of congenital cataract. METHODS: Protein-protein interactions were determined by confocal fluorescence resonance energy transfer (FRET) microscopy using green fluorescence protein (GFP) as the donor and red fluorescence protein (RFP) as the acceptor. The lens vimentin gene was fused into a GFP vector and the alphaB-crystallin (WT or R120G mutant) gene was fused into the RFP vector. The donor-acceptor plasmid pairs of intermediate filament (IF)-GFP and alphaB-RFP were co-transfected into HeLa cells. After incubation, confocal fluorescence images of the transfected cells were taken. FRET was estimated by the acceptor photobleaching method. Protein-protein interaction was evaluated by FRET efficiency. RESULTS: The confocal fluorescence images showed that the cells expressing vimentin and R120G alphaB-crystallin contained large amounts of protein aggregates while few vimentin fibers were observed. FRET efficiency analyses indicated that vimentin had a significantly greater protein-protein interaction with R120G alphaB-crystallin than with WT alphaB-crystallin. CONCLUSIONS: Our results show that the R120G alphaB-crystallin mutant promoted vimentin aggregation through increased protein-protein interaction. This process may contribute to the formation of congenital cataract.

Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells.

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only alpha-crystallin or some specific gamma-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (alphaA-, alphaB-, betaB2-, or gammaC-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from alphaA- and alphaB-crystallin to betaB2- and gammaC-crystallin. Competitive interaction study using untagged alphaA-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and alphaA-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm.

Degradation of C-terminal truncated alpha A-crystallins by the ubiquitin-proteasome pathway.

PURPOSE: Calpain-mediated C-terminal cleavage of alpha A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated alpha A-crystallins. METHODS: Recombinant wild-type (wt) alpha A-crystallin and C-terminal truncated alpha A(1-168)-, alpha A(1-163)-, and alpha A(1-162)-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated alpha A-crystallins were labeled with (125)I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe Bis-ANS were used to characterize the wt and truncated alpha A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering. RESULTS: Whereas wt alpha A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, alpha A(1-168)-crystallin was resistant to degradation. The susceptibility of alpha A(1-163)-crystallin to degradation was comparable to that of wt alpha A-crystallin. However, alpha A(1-162)-crystallin was much more susceptible than wt alpha A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated alpha A(1-162)-crystallins requires adenosine triphosphate (ATP) and was stimulated by addition of a ubiquitin-conjugating enzyme, Ubc4. The degradation was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative mutant of ubiquitin, K6W-Ub, indicating that at least part of the proteolysis was mediated by the UPP. Spectroscopic analyses of wt and C-terminal truncated alpha A-crystallins revealed that C-terminal truncation of alpha A-crystallin resulted in only subtle changes in secondary structures. However, C-terminal truncations resulted in significant changes in surface hydrophobicity and thermal stability. Thus, these conformational changes may reveal or mask the signals for the ubiquitin-dependent degradation. CONCLUSIONS: The present data demonstrate that C-terminal cleavage of alpha A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of alpha A(1-162) by the UPP may prevent its accumulation in the lens.

Protein-protein interactions among human lens acidic and basic beta-crystallins.

Human lens beta-crystallin contains four acidic (betaA1-->betaA4) and three basic (betaB1-->betaB3) subunits. They oligomerize in the lens, but it is uncertain which subunits are involved in the oligomerization. We used a two-hybrid system to detect protein-protein interactions systematically. Proteins were also expressed for some physicochemical studies. The results indicate that all acidic-basic pairs (betaA-betaB) except betaA4-betaBs pairs show strong hetero-molecular interactions. For acidic or basic pairs, only two pairs (betaA1-betaA1 and betaA3-betaA3) show strong self-association. betaA2 and betaA4 show very weak self-association, which arises from their low solubility. Confocal fluorescence microscopy shows enormous protein aggregates in betaA2- or betaA4-crystallin transfected cells. However, coexpression with betaB2-crystallin decreased both the number and size of aggregates. Circular dichroism indicates subtle differences in conformation among beta-crystallins that may have contributed to the differences in interactions.