[Focal segmental glomerulosclerosis (FSGS). Molecular defects and clinical relevance].
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Adaptor Proteins, Signal Transducing↗
Biomedical subjects
M Schiffer
Publications and source records attributed to M Schiffer.
Clinal variation and laboratory adaptation in the rainforest species Drosophila birchii for stress resistance, wing size, wing shape and development time.
Clinal variation has been described in many invertebrates including drosophilids but usually over broad geographical gradients. Here we describe clinal variation in the rainforest species Drosophila birchii from Queensland, Australia, and potential confounding effects of laboratory adaptation. Clinal variation was detected for starvation and development time, but not for size or resistance to temperature extremes. Starvation resistance was higher at southern locations. Wing shape components were not associated with latitude although they did differ among populations. Time in laboratory culture did not influence wing size or heat knockdown resistance, but increased starvation resistance and decreased recovery time following a cold shock. Laboratory culture also increased development time and altered wing shape. The results indicate that clinal patterns can be detected in Drosophila over a relatively narrow geographical area. Laboratory adaptation is unlikely to have confounded the detection of geographical patterns.
Adaptation, Physiological↗
Genome-scale expression of proteins from Bacillus subtilis.
We have applied high throughput methods for cloning and expression of more than 850 genes from the Bacillus subtilis genome. The process uses 96-well plates and is automated from the level of primer design to the detection of soluble protein by a tag detection screen. This process was applied to a set of cytoplasmic targets from Bacillus subtilis to produce clones expressing soluble protein for incorporation into the structure determination pipeline of the Midwest Center for Structural Genomics. We also evaluated the feasibility of these plate-based methods for domain-based cloning and expression of secretory proteins and putative soluble domains of membrane proteins. This approach shows promise for implementation in a high throughput format and could provide additional target resources for structure determination. The continued development of new technologies that can be implemented in an automated format will be essential for continued success in the structural genomic programs.
Bacillus subtilis↗
Molecular, morphological and behavioural data reveal the presence of a cryptic species in the widely studied Drosophila serrata species complex.
The Drosophila serrata species complex from Australia and New Guinea has been widely used in evolutionary studies of speciation and climatic adaptation. It is believed to consist of D. serrata, D. birchii and D. dominicana, although knowledge of the latter is limited. Here we present evidence for a previously undescribed cryptic member of the D. serrata species complex. This new cryptic species is widespread in far north Queensland, Australia and is likely to have been previously mistaken for D. serrata. It shows complete reproductive isolation when crossed with both D. serrata and D. birchii. The cryptic species can be easily distinguished from D. serrata and D. birchii using either microsatellite loci or visual techniques. Although it occurs sympatrically with both D. serrata and D. birchii, it differs from these species in development time, viability, wing size and wing morphology. Its discovery explains patterns of recently described mitochondrial DNA divergence within D. serrata, and may also help to clarify some ambiguities evident in early evolutionary literature on reproductive incompatibility within the D. serrata species complex.
Analysis of Variance↗
Low potential for climatic stress adaptation in a rainforest Drosophila species.
The ability of sensitive rainforest species to evolve in response to climate change is largely unknown. We show that the Australian tropical rainforest fly Drosophila birchii exhibits clinal variation in desiccation resistance, but the most resistant population lacks the ability to evolve further resistance even after intense selection for over 30 generations. Parent-offspring comparisons indicate low heritable variation for this trait but high levels of genetic variation for morphology. D. birchii also exhibits abundant genetic variation at microsatellite loci. The low potential for resistance evolution highlights the importance of assessing evolutionary potential in targeted ecological traits and species from threatened habitats.
Adaptation, Physiological↗
The structure of a mutant photosynthetic reaction center shows unexpected changes in main chain orientations and quinone position.
We report on the unexpected structural changes caused by substitution of acidic amino acids in the Q(B) binding pocket of the bacterial photosynthetic reaction center by alanines. The mutations targeted key residues L212Glu and L213Asp of this transmembrane protein-cofactor complex. The amino acid substitutions in the L212Ala-L213Ala mutant reaction center ("AA") were known to affect the delivery of protons after the light-induced generation of Q(B)(-), which renders the AA strain incapable of photosynthetic growth. The AA structure not only revealed side chain rearrangements but also showed movement of the main chain segments that are contiguous with the mutation sites. The alanine substitutions caused an expansion of the cavity rather than its collapse. In addition, Q(B) is found mainly in the binding site that is proximal to the iron-ligand complex (closest to Q(A)) as opposed to its distal binding site (furthest from Q(A)) in the structure of the wild-type reaction center. The observed rearrangements in the structure of the AA reaction center establish a new balance between charged residues of an interactive network near Q(B). This structurally and electrostatically altered complex forms the basis for future understanding of the structural basis for proton transfer in active reaction centers which retain the alanine substitutions but carry a distant compensatory mutation.
Amino Acid Substitution↗
Increasing protein stability by polar surface residues: domain-wide consequences of interactions within a loop.
We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein's function.
Amino Acid Sequence↗
Factors contributing to decreased protein stability when aspartic acid residues are in beta-sheet regions.
Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.
Aspartic Acid↗
Revealing the involvement of extended hydrogen bond networks in the cooperative function between distant sites in bacterial reaction centers.
In reaction center proteins of photosynthetic bacteria, the amplitude of proton uptake induced by the one-electron reduction of either of the two quinone electron acceptors (Q(A) and Q(B)) is an intrinsic observable of the electrostatic interactions associated with the redox function of the complex. We report here that, in Rhodobacter capsulatus, complete restoration of proton uptake (upon formation of Q(A)(-) and Q(B)(-)) to the level found in the wild type is observed in a mutant reaction center in which a tyrosine substitution in the Q(A) environment (Ala(M274) --> Tyr) is coupled with mutations of acidic residues near Q(B) (Glu(L212) --> Ala/Asp(L213) --> Ala) that initially cancel the proton uptake above pH 8. This result demonstrates that proton uptake occurs by strong cooperation between structural motifs, such as hydrogen-bonded networks, that span the 18 A distance between the two quinone acceptors.
Hydrogen Bonding↗
Trapping reactions of an intermediate containing a tungsten-phosphorus triple bond with alkynes.
The thermolysis of the phosphinidene complex [Cp*P[W(CO)5]2] (1) in toluene in the presence of tBuC(triple bond)CMe leads to the four-membered ring complexes [[[eta2-C(Me)C(tBu)]Cp*(CO)W(mu3-P)[W(CO)3]][eta4:eta1:eta1-P[W(CO)5]WCp*(CO)C(Me)C(tBu)]] (4) as the major product and [[W[Cp*(CO)2]W(CO)2WCp*(CO)[eta1:eta1-C(Me)C(tBu)]](mu,eta3:eta2:eta1-P2[W(CO)5]] (5). The reaction of 1 with PhC(triple bond)CPh leads to [[W(Co)2[eta2-C(Ph)C(Ph)]][(eta4:eta1-P(W(CO)5]W[Cp*(CO)2)C(Ph)C(Ph)]] (6). The products 4 and 6 can be regarded as the formal cycloaddition products of the phosphido complex intermediate [Cp*(CO)2W(triple bond)P --> W(CO)5] (B), formed by Cp* migration within the phosphinidene complex 1. Furthermore, the reaction of 1 with PhC(triple bond)CPh gives the minor product [[[eta2:eta1-C(Ph)C(Ph)]2[W(CO)4]2][mu,eta1:eta1-P[C(Me)[C(Me)]3C(Me)][C(Ph)](C(Ph)]] (7) as a result of a 1,3-dipolaric cycloaddition of the alkyne into a phosphaallylic subunit of the Cp*P moiety of 1. Compounds 4-7 have been characterized by means of their spectroscopic data as well as by single-crystal X-ray structure analysis.
Journal Article↗
Apoptosis in podocytes induced by TGF-beta and Smad7.
Primary and secondary forms of focal segmental glomerulosclerosis (FSGS) are characterized by depletion of podocytes and constitute a central manifestation of chronic progressive glomerular diseases. Here we report that podocytes undergo apoptosis at early stages in the course of progressive glomerulosclerosis in TGF-beta1 transgenic mice. Apoptosis is associated with progressive depletion of podocytes and precedes mesangial expansion. Smad7 protein expression is strongly induced specifically in damaged podocytes of transgenic mice and in cultured murine podocytes treated with TGF-beta. TGF-beta1 and Smad7 each induce apoptosis in podocytes, and their coexpression has an additive effect. Activation of p38 MAP kinase and caspase-3 is required for TGF-beta-mediated apoptosis, but not for apoptosis induced by Smad7. Unlike TGF-beta, Smad7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NF-kappaB. Our results suggest a novel functional role for Smad7 as amplifier of TGF-beta-induced apoptosis in podocytes and a new pathomechanism for podocyte depletion in progressive glomerulosclerosis.
Animals↗
Protein conformation and disease: pathological consequences of analogous mutations in homologous proteins.
The antibody light chain variable domain (V(L))(1) and myelin protein zero (MPZ) are representatives of the functionally diverse immunoglobulin superfamily. The V(L) is a subunit of the antigen-binding component of antibodies, while MPZ is the major membrane-linked constituent of the myelin sheaths that coat peripheral nerves. Despite limited amino acid sequence homology, the conformations of the core structures of the two proteins are largely superimposable. Amino acid variations in V(L) account for various conformational disease outcomes, including amyloidosis. However, the specific amino acid changes in V(L) that are responsible for disease have been obscured by multiple concurrent primary structure alterations. Recently, certain demyelination disorders have been linked to point mutations and single amino acid polymorphisms in MPZ. We demonstrate here that some pathogenic variations in MPZ correspond to changes suspected of determining amyloidosis in V(L). This unanticipated observation suggests that studies of the biophysical origin of conformational disease in one member of a superfamily of homologous proteins may have implications throughout the superfamily. In some cases, findings may account for overt disease; in other cases, due to the natural repertoire of inherited polymorphisms, variations in a representative protein may predict subclinical impairment of homologous proteins.
Amino Acid Sequence↗
Modeling the effects of mutations on the free energy of the first electron transfer from QA- to QB in photosynthetic reaction centers.
Numerical calculations of the free energy of the first electron transfer in genetically modified reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus were carried out from pH 5 to 11. The multiconformation continuum electrostatics (MCCE) method allows side chain, ligand, and water reorientation to be embedded in the calculations of the Boltzmann distribution of cofactor and amino acid ionization states. The mutation sites whose effects have been modeled are L212 and L213 (the L polypeptide) and two in the M polypeptide, M43(44) and M231(233) in Rb. capsulatus (Rb. sphaeroides). The results of the calculations were compared to the experimental data, and very good agreement was found especially at neutral pH. Each mutation removes or introduces ionizable residues, but the protein maintains a net charge close to that in native RCs through ionization changes in nearby residues. This reduces the effect of mutation and makes the changes in state free energy smaller than would be found in a rigid protein. The state energy of QA-QB and QAQB- states have contributions from interactions among the residues as well as with the quinone which is ionized. For example, removing L213Asp, located in the QB pocket, predominantly changes the free energy of the QA-QB state, where the Asp is ionized in native RCs rather than the QAQB- state, where it is neutral. Side chain, hydroxyl, and water rearrangements due to each of the mutations have also been calculated showing water occupancy changes during the QA- to QB electron transfer.
Alanine↗
Pnictides as symmetrically bridging ligands in novel neutral complexes
The reaction of [LWCl] (3) [L = N(CH2CH2NiPr)3] with LiE(SiMe3)2 (E = P, As, Sb) yields the novel, neutral pnictido-bridged complexes [LW = E = WL] (5-7). By following the reaction, which starts from the LiP-(SiMe3)2 derivative, by 31P NMR spectroscopy, the formation of an intermediate with a terminal pnictido ligand can be ruled out. The paramagnetic complexes 5-7 are comprehensively spectroscopically characterised. The X-ray structure analysis of the heterocumulenes 5-7 reveals a linear structure in which the two W-"tren" units bind to the central pnictido atom in a staggered conformation ["tren" = tren-based ligand; tren = tris(2-aminoethyl)-amine. When N2 is used as the inert gas in the synthesis of the starting material [N(CH2CH2NNp)3WCl] [Np = CH2C-(CH3)3], the complex [[N(CH2CH2NNp)3]W2(mu, eta 1: eta 1-N2)] (4) is formed as a side product. Complex 4 possesses a hydrazido(4-) (N2(4-)) ligand connected by two tungsten-"tren" moieties.
Journal Article↗
Smad proteins and transforming growth factor-beta signaling.
It is now generally accepted that transforming growth factor-beta (TGF-beta) has an important role in the pathogenesis of both acute and chronic forms of renal disease. Although TGF-beta's potent fibrogenic activity is considered a major factor in chronic progression of renal disease, this cytokine participates in the control of several fundamental cellular responses in the kidney including inflammation, programmed cell death, cell growth, cell differentiation, and cellular hypertrophy. Recent identification of Smad proteins as intracellular mediators of TGF-beta signaling has provided important insights into mechanisms that may determine the specificity of TGF-beta action in different renal and inflammatory cells. Thus, Smads are characterized by an astonishingly complex array of molecular and functional interactions with other signaling pathways. These emerging patterns of signaling cross talk involving Smad proteins suggest a dynamic profile of positive or negative transmodulation of TGF-beta signaling, depending on the cellular context. Understanding the interplay between these signaling cascades is an important field of investigation that will ultimately reveal new targets for precise and selective modulation of TGF-beta's diverse actions in renal diseases.
Animals↗
Change in dimerization mode by removal of a single unsatisfied polar residue located at the interface.
The importance of unsatisfied hydrogen bonding potential on protein-protein interaction was studied. Two alternate modes of dimerization (conventional and flipped form) of an immunoglobulin light chain variable domain (V(L)) were previously identified. In the flipped form, interface residue Gln89 would have an unsatisfied hydrogen bonding potential. Removal of this Gln should render the flipped dimer as the more favorable quaternary form. High resolution crystallographic studies of the Q89A and Q89L mutants show, as we predicted, that these proteins indeed form flipped dimers with very similar interfaces. A small cavity is present in the Q89A mutant that is reflected in the approximately 100 times lower association constant than found for the Q89L mutant. The association constant of Q89A and Q89L proteins (4 x 10(6) M(-1) and >10(8) M(-1)) are 10- and 1,000-fold higher than that of the wild-type protein that forms conventional dimers clearly showing the energetic reasons for the flipped dimer formation.
Crystallography, X-Ray↗
Proton uptake by bacterial reaction centers: the protein complex responds in a similar manner to the reduction of either quinone acceptor.
In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone Q(A)(-) and the secondary acceptor Q(B)(-) after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near Q(B); some of them carry additional mutations distant from the quinone sites (M231Arg --> Leu, M43Asn --> Asp, M5Asn --> Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala --> Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either Q(A)(-) or Q(B)(-), suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to Q(B) is evident from examination of the pattern of H(+)/Q(A)(-) proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q(-) species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.
Hydrogen-Ion Concentration↗
Few clues for AIDS vaccines from structural data on gp120 and its receptors and antibodies.
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AIDS Vaccines↗