The circuitry of cargo flux in the ESCRT pathway

Meiosis-specific mammalian cohesin SMC1β is required for complete sister chromatid cohesion and proper axes/loop structure of axial elements (AEs) and synaptonemal complexes (SCs). During prophase I, telomeres attach to the nuclear envelope (NE), but in Smc1β^−/− meiocytes, one fifth of their telomeres fail to attach. This study reveals that SMC1β serves a specific role at telomeres, which is independent of its role in determining AE/SC length and loop extension. SMC1β is necessary to prevent telomere shortening, and SMC3, present in all known cohesin complexes, properly localizes to telomeres only if SMC1β is present. Very prominently, telomeres in Smc1β^−/− spermatocytes and oocytes loose their structural integrity and suffer a range of abnormalities. These include disconnection from SCs and formation of large telomeric protein–DNA extensions, extended telomere bridges between SCs, ring-like chromosomes, intrachromosomal telomeric repeats, and a reduction of SUN1 foci in the NE. We suggest that a telomere structure protected from DNA rearrangements depends on SMC1β.     

Selective deletion of forebrain glycogen synthase kinase 3�� reveals a central role in serotonin-sensitive anxiety and social behaviour

Serotonin (5-HT) neurotransmission is thought to underlie mental illnesses, such as bipolar disorder, depression, autism and schizophrenia. Independent studies have indicated that 5-HT or drugs acting on 5-HT neurotransmission regulate the serine/threonine kinase glycogen synthase kinase 3β (GSK3β). Furthermore, GSK3β inhibition rescues behavioural abnormalities in 5-HT-deficient mice with a loss-of-function mutation equivalent to the human variant (R441H) of tryptophan hydroxylase 2. In an effort to define neuroanatomical correlates of GSK3β activity in the regulation of behaviour, we generated CamKIIcre-floxGSK3β mice in which the gsk3b gene is postnatally inactivated in forebrain pyramidal neurons. Behavioural characterization showed that suppression of GSK3β in these brain areas has anxiolytic and pro-social effects. However, while a global reduction of GSK2β expression reduced responsiveness to amphetamine and increased resilience to social defeat, these behavioural effects were not found in CamKIIcre-floxGSK3β mice. These findings demonstrate a dissociation of behavioural effects related to GSK3 inhibition, with forebrain GSK3β being involved in the regulation of anxiety and sociability while social preference, resilience and responsiveness to psychostimulants would involve a function of this kinase in subcortical areas such as the hippocampus and striatum.     

MutS�� and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells

Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington’s disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutSβ (MSH2–MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (∼30–40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutSβ subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutSα subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutSβ, but not MutSα, was enriched at the TNR. These findings imply a direct role for MutSβ in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutSβ, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.     

Effective Size of Populations under Selection

Equations to approximate the effective size (N(e)) of populations under continued selection are obtained that include the possibility of partial full-sib mating and other systems such as assortative mating. The general equation for the case of equal number of sexes and constant number of breeding individuals (N) is N(e) = 4N/[2(1 - α(I)) + (S(k)(2) + 4Q(2)C(2)) (1 + α(I) + 2α(O))], where S(k)(2) is the variance of family size due to sampling without selection, C(2) is the variance of selective advantages among families (the squared coefficient of variation of the expected number of offspring per family), α(I) is the deviation from Hardy-Weinberg proportions, α(O) is the correlation between genes of male and female parents, and Q(2) is the term accounting for the cumulative effect of selection on an inherited trait. This is obtained as Q = 2/[2 - G(1 + r)], where G is the remaining proportion of genetic variance in selected individuals and r is the correlation of the expected selective values of male and female parents. The method is also extended to the general case of different numbers of male and female parents. The predictive value of the formulae is tested under a model of truncation selection with the infinitesimal model of gene effects, where C(2) and G are a function of the selection intensity, the heritability and the intraclass correlation of sibs. Under random mating r = α(I) = -1/(N - 1) and α(O) = 0. Under partial full-sib mating with an average proportion β of full-sib matings per generation, r & β and α(O) & α(I) & β/ (4 - 3β). The prediction equation is compared to other approximations based on the long-term contributions of ancestors to descendants. Finally, based on the approach followed, a system of mating (compensatory mating) is proposed to reduce rates of inbreeding without loss of response in selection programs in which selected individuals from the largest families are mated to those from the smallest families.     

Intrinsic α‐helical and β‐sheet conformational preferences: A computational case study of alanine

A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle (-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (N–C_α–C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the “bridge” region of-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between -ψ conformations and τ.     

αA-Crystallin-Derived Mini-Chaperone Modulates Stability and Function of Cataract Causing αAG98R-Crystallin

Background

A substitution mutation in human αA-crystallin (αAG98R) is associated with autosomal dominant cataract. The recombinant mutant αAG98R protein exhibits altered structure, substrate-dependent chaperone activity, impaired oligomer stability and aggregation on prolonged incubation at 37°C. Our previous studies have shown that αA-crystallin–derived mini-chaperone (DFVIFLDVKHFSPEDLTVK) functions like a molecular chaperone by suppressing the aggregation of denaturing proteins. The present study was undertaken to determine the effect of αA-crystallin–derived mini-chaperone on the stability and chaperone activity of αAG98R-crystallin.

Methodology/Principal Findings

Recombinant αAG98R was incubated in presence and absence of mini-chaperone and analyzed by chromatographic and spectrometric methods. Transmission electron microscope was used to examine the effect of mini-chaperone on the aggregation propensity of mutant protein. Mini-chaperone containing photoactive benzoylphenylalanine was used to confirm the interaction of mini-chaperone with αAG98R. The rescuing of chaperone activity in mutantα-crystallin (αAG98R) by mini-chaperone was confirmed by chaperone assays. We found that the addition of the mini-chaperone during incubation of αAG98R protected the mutant crystallin from forming larger aggregates that precipitate with time. The mini-chaperone-stabilized αAG98R displayed chaperone activity comparable to that of wild-type αA-crystallin. The complexes formed between mini-αA–αAG98R complex and ADH were more stable than the complexes formed between αAG98R and ADH. Western-blotting and mass spectrometry confirmed the binding of mini-chaperone to mutant crystallin.

Conclusion/Significance

These results demonstrate that mini-chaperone stabilizes the mutant αA-crystallin and modulates the chaperone activity of αAG98R. These findings aid in our understanding of how to design peptide chaperones that can be used to stabilize mutant αA-crystallins and preserve the chaperone function.     

GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a ��-Arrestin 1-mediated ERK1/2 Activation in Pancreatic ��-Cells

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gα_s/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacological inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.     

Congenital cataract causing mutants of αA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway

Background

Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy.

Methodology/Principal Findings

YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control.

Conclusions/Significance

Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.     

Mutations in human αA-crystallin/sHSP affect subunit exchange interaction with αB-crystallin

Background

Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.

Methodology/Principal Findings

In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.

Conclusions/Significance

Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.     

Phosphorylation of Synucleins by Members of the Polo-like Kinase Family

Phosphorylation of α-synuclein (α-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating α-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate α-syn and β-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate α-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate β-syn at Ser-118, whereas no phosphorylation of γ-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103–140) of α-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of α-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) α-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in α-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of α-syn and β-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.     

The Direct Binding of Insulin-like Growth Factor-1 (IGF-1) to Integrin ��v��3 Is Involved in IGF-1 Signaling

It has been proposed that ligand occupancy of integrin αvβ3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of αvβ3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that αvβ3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of αvβ3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin αvβ3 plays a role in IGF-1 signaling through ternary complex formation (αvβ3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.Integrins are a family of cell adhesion receptors that mediate cell-extracellular matrix (ECM)³ interaction and cell-cell interaction (1). It has been proposed that signaling from inside the cells regulates the ligand binding affinity of integrins (inside-out signaling) (2). Each integrin is a heterodimer containing α and β subunits. At present 18 α and 8 β subunits have been identified that combine to form 24 integrins (3).It has been reported that integrin αvβ3 plays a role in cancer proliferation and invasiveness. High levels of integrin αvβ3 correlate with growth and/or progression of melanoma (4, 5), neuroblastoma (6), breast cancer (7, 8), colon cancer (9), ovarian cancer (10), and cervical cancer (11). Moreover, individuals homozygous for the β3L33P polymorphism that enhances the ligand binding affinity of β3 integrins have an increased risk to develop breast cancer, ovarian cancer, and melanoma (12). However, it remains unclear whether and how increased levels of αvβ3 on tumor cells contribute to cancer development.Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone (75 kDa) that has a high degree of structural similarity to human proinsulin. IGF-1 acts through binding to the type I IGF receptor (IGF1R), a receptor tyrosine kinase. The IGF1R is a heterotetramer that consists of two α-subunits that contain the ligand-binding domains and two β-subunits that contain the tyrosine kinase activity. After ligand binding, the receptor undergoes a conformational change resulting in the activation of the tyrosine kinase, which results in transphosphorylation of the opposite β-subunit on specific tyrosine residues. These phosphotyrosines then bind to adapter molecules such as Shc and IRS-1. Phosphorylation of these proteins leads to activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) signaling pathways (reviewed in Ref. 13).IGF-1 has been implicated in cancer progression (14). One of the major actions of IGF-1 is to inhibit apoptosis. IGF-1 confers resistance to chemotherapy and radiation therapy. IGF-1 expression levels are increased in breast, lung, prostate, and many other cancers. Several strategies to target IGF-1 signaling have been extensively studied, including small interfering RNA and monoclonal antibodies for IGF1R and kinase inhibitors to inhibit the enzymatic activity of the receptor. The IGF-1 system is a therapeutic target for cancer, and elucidation of the IGF-1 signaling pathway should have a major impact in designing new therapeutic strategies.It has been proposed that ligand occupancy of αvβ3 with ECM ligands such as vitronectin plays a critical role in enhancing IGF-1 signaling (14). It has been reported that inhibiting αvβ3-ECM interaction (“ligand occupancy”) of αvβ3 inhibited IGF-1 actions selectively in cell types that express αvβ3 (14). Inhibiting ligand occupancy of αvβ3 blocked IGF-1-induced cell migration (15), DNA synthesis, IRS-1 phosphorylation, and IGF1R-linked downstream signaling events, such as activation of phosphatidylinositol 3-kinase and ERK1/2 (16).In the present study, we demonstrated that expression of αvβ3 enhanced proliferation of ovarian cancer cells in the presence of fetal bovine serum (FBS) and in serum-free conditions if IGF-1 was present. This suggests that IGF-1 is involved in enhanced proliferation of αvβ3-expressing cells. We demonstrated that αvβ3 bound to IGF-1 in several different binding assays. We found that two Arg residues at positions 36 and 37 in the C-domain of IGF-1 are critical for integrin binding by docking simulation and mutagenesis. Mutation of these Arg residues to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, the R36E/R37E mutant was defective in inducing cell proliferation and IGF-1 intracellular signaling, although it still bound to IGF1R. We demonstrated that wild type IGF-1 mediated co-precipitation of αvβ3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 plays a role in IGF-1 signaling.     

The effect of finasteride and dutasteride on the growth of WPE1-NA22 prostate cancer xenografts in nude mice

Background

5α-reductase 1 (5αR1) and 5α-reductase 2 (5αR2) convert testosterone into the more potent androgen dihydrotestosterone. 5αR2 is the main isoenzyme in normal prostate tissue; however, most prostate tumors have increased 5αR1 and decreased 5αR2 expression. Previously, finasteride (5αR2 inhibitor) treatment begun 3 weeks post-tumor implantation had no effect on Dunning R3327-H rat prostate tumor growth. We believe the tumor compensated for finasteride treatment by increasing tumor 5αR1 expression or activity. We hypothesize that finasteride treatment would not significantly alter tumor growth even if begun before tumor implantation, whereas dutasteride (5αR1 and 5αR2 inhibitor) treatment would decrease tumor growth regardless of whether treatment was initiated before or after tumor implantation.

Methodology/Principal Findings

Sixty 8-week-old male nude mice were randomized to Control, Pre- and Post-Finasteride, and Pre- and Post-Dutasteride (83.3 mg drug/kg diet) diet groups. Pre- and post-groups began their treatment diets 1–2 weeks prior to or 3 weeks after subcutaneous injection of 1×10⁵ WPE1-NA22 human prostate cancer cells, respectively. Tumors were allowed to grow for 22 weeks; tumor areas, body weights, and food intakes were measured weekly. At study''s conclusion, prostate and seminal vesicle weights were significantly decreased in all treatment groups versus the control; dutasteride intake significantly decreased seminal vesicle weights compared to finasteride intake. No differences were measured in final tumor areas or tumor weights between groups, likely due to poor tumor growth. In follow-up studies, proliferation of WPE1-NA22 prostate cancer cells and parent line RWPE-1 prostate epithelial cells were unaltered by treatment with testosterone, dihydrotestosterone, or mibolerone, suggesting that these cell lines are not androgen-sensitive.

Conclusion

The lack of response of WPE1-NA22 prostate cancer cells to androgen treatment may explain the inadequate tumor growth observed. Additional studies are needed to determine whether finasteride and dutasteride are effective in decreasing prostate cancer development/growth.     

The Novel S527F Mutation in the Integrin ��3 Chain Induces a High Affinity ��IIb��3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor αIIbβ3 in a patient with an ill defined platelet disorder: one in the β3 gene (S527F) and two in the αIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant αIIbβ3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the β3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated αIIbβ3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of αIIbβ3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe⁵²⁷ in β3 and the calf-1 domain in αIIb and by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin receptors that consist of noncovalently linked α/β-heterodimers. They are cell-surface receptors that play a role in cell-cell and cell-matrix interactions. Under resting conditions, integrin receptors adopt the low affinity conformation and do not interact with their ligands. Inside-out signaling turns the receptor into a high affinity conformation capable of ligand binding. Ligand binding itself induces additional conformational changes resulting in exposure of neoantigenic sites called ligand-induced binding sites (LIBS)³ and generates in turn outside-in signaling, which triggers a range of downstream signals (1, 2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In flowing blood under resting conditions, αIIbβ3 does not interact with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere at sites of vascular injury and become activated. As a consequence, αIIbβ3 adopts the high affinity conformation and binds fibrinogen. This results in platelet aggregation and thrombus formation, which eventually will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal ligand-binding head domain (the β-propeller domain in the αIIb chain and the βI domain in the β3 chain) standing on two long legs or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial growth factor-like (I-EGF), and β-tail domains in the β3 chain), followed by transmembrane and cytoplasmic domains (1, 2). X-ray crystal structures of the extracellular domain of non-activated αVβ3 revealed that the legs are severely bent, putting the head domain next to the membrane-proximal portions of the legs (4, 5). The bending occurs between I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1 domains in the α-subunit. This bent conformation represents the low affinity state of the receptor. The high affinity state of the receptor is induced by activation and is associated with a large-scale conformational rearrangement in which the integrin extends with a switchblade-like motion (2). Recently, the crystal structure of the entire extracellular domain of αIIbβ3 in its low affinity conformation was resolved and revealed that this integrin also adopts the bent conformation under resting conditions (6). Structural rearrangements in αIIbβ3 between the bent and extended conformations are similar to what has been reported for other integrins (7).We report here that the S527F mutation in the I-EGF3 region of the β3 polypeptide chain of the αIIbβ3 receptor induces a constitutively active receptor adopting an extended high affinity conformation. This was evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding. These data were further corroborated by modeling the replacement of Ser⁵²⁷ with Phe in the crystal structure of the extracellular domain of αIIbβ3. In this model, the S527F mutation decreases the flexibility of I-EGF3 and appears to prevent movement of the lower β-leg into the cleft between the upper β-leg and the lower α-leg. As a consequence, formation of the bent conformation of the non-activated receptor is hampered.     

Biodiversity Patterns along Ecological Gradients: Unifying β-Diversity Indices

Ecologists have developed an abundance of conceptions and mathematical expressions to define β-diversity, the link between local (α) and regional-scale (γ) richness, in order to characterize patterns of biodiversity along ecological (i.e., spatial and environmental) gradients. These patterns are often realized by regression of β-diversity indices against one or more ecological gradients. This practice, however, is subject to two shortcomings that can undermine the validity of the biodiversity patterns. First, many β-diversity indices are constrained to range between fixed lower and upper limits. As such, regression analysis of β-diversity indices against ecological gradients can result in regression curves that extend beyond these mathematical constraints, thus creating an interpretational dilemma. Second, despite being a function of the same measured α- and γ-diversity, the resultant biodiversity pattern depends on the choice of β-diversity index. We propose a simple logistic transformation that rids beta-diversity indices of their mathematical constraints, thus eliminating the possibility of an uninterpretable regression curve. Moreover, this transformation results in identical biodiversity patterns for three commonly used classical beta-diversity indices. As a result, this transformation eliminates the difficulties of both shortcomings, while allowing the researcher to use whichever beta-diversity index deemed most appropriate. We believe this method can help unify the study of biodiversity patterns along ecological gradients.     

Role of α-Factor and the Mfα1 α-Factor Precursor in Mating in Yeast

The peptide pheromones secreted by a and α cells (called a-factor and α-factor, respectively) are each encoded by two structural genes. For strains of either mating type, addition of exogenous pheromone does not alleviate the mating defect of mutants with disruptions of both structural genes. In addition, a particular insertion mutation in the major α-factor structural gene (MFα1) that should result in an altered product inhibits α mating. These results suggested that the pheromone precursors (the MFα1 pro region in particular) might play a second role in mating separate from the role of pheromone production. To analyze the role of α-factor and the MFα1 precursor in α mating, we have constructed two classes of mutants. The mating defects of mutants that should produce the MFα1 pro region peptide but no α-factor could not be alleviated by addition of exogenous α-factor in crosses to a wild-type a strain, indicating that the previous results were not due to an inability of the disruption mutants to produce the pro region peptide. Mutants able to produce α-factor, but with a variety of alterations in MFα1 precursor structure, mated at levels proportional to the levels of α-factor produced, suggesting that the only role of the α-factor precursor in mating is to produce α-factor. Both of these results argue against a role for the MFα1 pro region separate from its role in α-factor production. We also describe results that show that in vivo production of α-factor'' (the form of α-factor encoded by one of the two α-factor repeats of MFα2) is equivalent to the major form of α-factor for induction of all responses necessary for mating. We discuss the implications of these results on the role of the pheromones in mating.     

Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants

Lens α-crystallin is an oligomeric protein with a molecular mass of 500–1000 kDa and a polydispersed assembly. It consists of two types of subunits, αA and αB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F αA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C αA, wild-type and R120G αB, wild-type and Q155* βB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C αA and R120G αB mutations despite conformational changes.