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M A Weiss

Publications and source records attributed to M A Weiss.

At least 91 records · Page 5Linked to original sources
PubMed1993

SRY recognizes conserved DNA sites in sex-specific promoters.

Formation of male-specific structures and regression of female primordia are regulated in early male embryogenesis by SRY, a single-copy gene on the Y chromosome. Assignment of SRY as the testis-determining factor in eutherian mammals is supported by molecular analysis of cytogenetic sex reversal (i.e., XX males and XY females) and by complementary studies of transgenic murine models. Here we characterize the putative DNA-binding domain of SRY, which contains a conserved sequence motif shared by high-mobility group nuclear proteins and a newly recognized class of transcription factors. The SRY DNA-binding domain specifically recognizes with nanomolar affinity proximal upstream elements (designated SRYe) in the promoters of the sex-specific genes encoding P450 aromatase and Mullerian inhibiting substance (MIS). P450 aromatase catalyzes the conversion of testosterone to estradiol, and in the male embryo its expression is down-regulated. Conversely, MIS is expressed in the male embryo to induce testicular differentiation and regression of female reproductive ducts. SRYe-binding activity is observed in nuclear extracts obtained from embryonic urogenital ridge immediately preceding morphologic testicular differentiation. Our results support the hypothesis that SRY directly controls male development through sequence-specific regulation of target genes.

Animals↗
PubMed1993

Paradoxical structure and function in a mutant human insulin associated with diabetes mellitus.

The solution structure of a diabetes-associated mutant human insulin (insulin Los Angeles; PheB24-->Ser) was determined by 13C-edited NMR spectroscopy and distance-geometry/simulated annealing calculations. Among vertebrate insulins PheB24 is invariant, and in crystal structures the aromatic ring appears to anchor the putative receptor-binding surface through long-range packing interactions in the hydrophobic core. B24 substitutions are of particular interest in relation to the mechanism of receptor binding. In one analogue ([GlyB24]insulin), partial unfolding of the B chain has been observed with paradoxical retention of near-native bioactivity. The present study of [SerB24]insulin extends this observation: relative to [GlyB24]insulin, near-native structure is restored despite significant loss of function. To our knowledge, our results provide the first structural study of a diabetes-associated mutant insulin and support the hypothesis that insulin undergoes a change in conformation on receptor binding.

Amino Acid Sequence↗
PubMed1993

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.

Amides↗
PubMed1993

Doppler ultrasound findings in reticular veins of the thigh subdermic lateral venous system and implications for sclerotherapy.

BACKGROUND: Treatment of varicose veins is directed towards eliminating sources of reflux causing venous hypertension in the superficial venous system. Doppler ultrasound is routinely used to localize sources of reflux prior to treatment of large varicose veins. OBJECTIVE: To assess the value of Doppler ultrasound in localizing reflux in subcutaneous reticular veins associated with groups of lateral thigh telangiectases. METHODS: Seven hundred patient records in which Doppler ultrasound was performed on thigh reticular veins were reviewed. Patients with reflux of the saphenofemoral junction, saphenopopliteal junction, or major saphenous system perforators were excluded. RESULTS: Audible reflux upon distal calf compression release was noted in thigh subcutaneous reticular veins of 618 out of 700 patients (88%). These incompetent reticular veins were associated with groups or webs of telangiectases and/or venulectases on the lateral thigh in almost all cases. Reflux was loudest just distal to the lateral femoral condyle. In 20 patients Doppler ultrasound was performed on a reticular vein while a telangiectasia associated with the reticular vein was injected with sclerosing solution. In 15 patients a clearly audible flow signal was heard in the reticular vein during injection. CONCLUSION: These findings indicate an association of telangiectatic veins with reticular veins on the thighs. Furthermore, this study underscores the value of Doppler in determining sources of reflux in smaller veins and implies that sclerotherapy of reticular veins is necessary for treatment of thigh telangiectatic webs.

Humans↗
PubMed1992

Nonlocal structural perturbations in a mutant human insulin: sequential resonance assignment and 13C-isotope-aided 2D-NMR studies of [PheB24-->Gly]insulin with implications for receptor recognition.

Insulin's mechanism of receptor binding is not well understood despite extensive study by mutagenesis and X-ray crystallography. Of particular interest are "anomalous" analogues whose bioactivities are not readily rationalized by crystal structures. Here the structure and dynamics of one such analogue (GlyB24-insulin) are investigated by circular dichroism (CD) and isotope-aided 2D-NMR spectroscopy. The mutant insulin retains near-native receptor-binding affinity despite a nonconservative substitution (PheB24-->Gly) in the receptor-binding surface. Relative to native insulin, GlyB24-insulin exhibits reduced dimerization; the monomer (the active species) exhibits partial loss of ordered structure, as indicated by CD studies and motional narrowing of selected 1H-NMR resonance. 2D-NMR studies demonstrate that the B-chain beta-turn (residues B20-23) and beta-strand (residues B24-B28) are destabilized; essentially native alpha-helical secondary structure (residues A3-A8, A13-A18, and B9-B19) is otherwise maintained. 13C-Isotope-edited NOESY studies demonstrate that long-range contacts observed between the B-chain beta-strand and the alpha-helical core in native insulin are absent in the mutant. Implications for the mechanism of insulin's interaction with its receptor are discussed.

Amino Acid Sequence↗
PubMed1992

Two-dimensional NMR studies of the zinc finger motif: solution structures and dynamics of mutant ZFY domains containing aromatic substitutions in the hydrophobic core.

Solution structures of mutant Zn fingers containing aromatic substitutions in the hydrophobic core are determined by 2D-NMR spectroscopy and distance-geometry/simulated annealing (DG/SA). The wild-type domain (designated ZFY-6) is derived from the human male-associated protein ZFY and represents a sequence motif (Cys-X2-Cys-X-Ar-X7-Leu-X2-His-X4-His) that differs from the consensus (Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-His-X3-His) in the location ("aromatic swap") and diversity (Ar = tyrosine, phenylalanine, or histidine) of the central aromatic residue (underlined). In a given ZFY domain the choice of a particular aromatic residue is invariant among vertebrates, suggesting that alternative "swapped" aromatic residues are functionally inequivalent. 2D-NMR studies of analogues containing tyrosine, phenylalanine, or histidine at the swapped site yield the following results. (i) The three DG/SA structures each retain the beta beta alpha motif and exhibit similar staggered-horizontal packing between the variant aromatic residue and the proximal histidine in the hydrophobic core. (ii) The structures and stabilities of the tyrosine and phenylalanine analogues are essentially identical, differing only by local exposure of polar (Tyr p-OH) or nonpolar (Phe p-H) surfaces. (iii) The dynamic stability of the histidine analogue is reduced as indicated by more rapid protein-deuterium exchange of hydrogen bonds related to secondary structure and amide-sulfur coordination (slowly exchanging amide resonances in D2O) and by more extensive averaging of main-chain dihedral angles (3J alpha NH coupling constants). An aspartic acid in the putative DNA recognition surface, whose configuration is well-defined as a possible helix N-cap in the tyrosine and phenylalanine analogues, exhibits multiple weak main-chain contacts in the NOESY spectrum of the histidine analogue; such NOEs are geometrically inconsistent and so provide complementary evidence for structural fluctuations. (iv) Because the three DG ensembles have similar apparent precision, the finding of reduced dynamic stability in the histidine analogue emphasizes the importance of experiments that directly probe fluctuations at several time scales. Our results provide insight into the design of biological metal-binding sites and the relationship of protein sequence to structure and dynamics.

Amino Acid Sequence↗
PubMed1992

Biochemical characterization of the Oct-2 POU domain with implications for bipartite DNA recognition.

B-cell specific regulation of immunoglobulin gene expression provides a model for the interaction of promoter and enhancer elements with eukaryotic sequence-specific DNA binding proteins. A critical element of this system, the octamer site (5'-ATGCAAAT-3'), is recognized by the B-cell transcription factor Oct-2. Octamer recognition is mediated by the POU domain, a conserved structural motif which--like the zinc finger and leucine zipper--defines a family of related transcription factors. Homologies among POU sequences suggest a bipartite structure, consisting of an N-terminal POU-specific subdomain and C-terminal variant homeodomain connected by a linker of variable length and sequence. As a first step toward a molecular understanding of the Oct-2 POU domain and its mechanism of DNA recognition, we have overexpressed in Escherichia coli the intact POU domain and subdomains as thrombin-cleavable fusion proteins and have purified these fragments to homogeneity following digestion with thrombin. Biochemical and biophysical characterization yields the following results. (i) The intact POU domain (166 residues) is monomeric and exhibits high-affinity octamer-specific DNA-binding activity. (ii) Limited proteolytic digestion demonstrates that the POU domain contains two proteolytically stable subdomains (the POU-specific subdomain and the variant homeodomain) connected by a proteolytically sensitive linker. (iii) The isolated subdomains are each monomeric and do not interact to form noncovalent heterodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Aromatic-aromatic interactions in the zinc finger motif. Analysis of the two-dimensional nuclear magnetic resonance structure of a mutant domain.

The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of pi-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromatic-aromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.

Amino Acid Sequence↗
PubMed1992

Structure and dynamics of des-pentapeptide-insulin in solution: the molten-globule hypothesis.

Structures of insulin in different crystal forms exhibit significant local and nonlocal differences, including correlated displacement of elements of secondary structure. Here we describe the solution structure and dynamics of a monomeric insulin analogue, des-pentapeptide-(B26-B30)-insulin (DPI), as determined by two-dimensional NMR spectroscopy and distance geometry/restrained molecular dynamics (DG/RMD). Although the solution structure of DPI exhibits a general similarity to its crystal structure, individual DG/RMD structures in the NMR ensemble differ by rigid-body displacements of alpha-helices that span the range of different crystal forms. These results suggest that DPI exists as a partially folded state formed by coalescence of distinct alpha-helix-associated microdomains. The physical reality of this model is investigated by comparison of the observed two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy (NOESY) spectrum with that predicted from crystal and DG/RMD structures. The observed NOESY spectrum contains fewer tertiary contacts than predicted by any single simulation, but it matches their shared features; such "ensemble correspondence" is likely to reflect the effect of protein dynamics on observed NOE intensities. We propose (i) that the folded state of DPI is analogous to that of a compact protein-folding intermediate rather than a conventional native state and (ii) that the molten state is the biologically active species. This proposal (the molten-globule hypothesis) leads to testable thermodynamic predictions and has general implications for protein design.

Calorimetry↗
PubMed1992

Mutations at the dimer, hexamer, and receptor-binding surfaces of insulin independently affect insulin-insulin and insulin-receptor interactions.

Mutagenesis of the dimer- and hexamer-forming surfaces of insulin yields analogues with reduced tendencies to aggregate and dramatically altered pharmacokinetic properties. We recently showed that one such analogue, HisB10----Asp, ProB28----Lys, LysB29----Pro human insulin (DKP-insulin), has enhanced affinity for the insulin receptor and is useful for studying the structure of the insulin monomer under physiologic solvent conditions [Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., & Shoelson, S. E. (1991) Biochemistry 30, 7373-7389]. DKP-insulin retains native secondary and tertiary structure in solution and may therefore provide an appropriate baseline for further studies of related analogues containing additional substitutions within the receptor-binding surface of insulin. To test this, we prepared a family of DKP analogues having potency-altering substitutions at the B24 and B25 positions using a streamlined approach to enzymatic semisynthesis which negates the need for amino-group protection. For comparison, similar analogues of native human insulin were prepared by standard semisynthetic methods. The DKP analogues show a reduced tendency to self-associate, as indicated by 1H-NMR resonance line widths. In addition, CD spectra indicate that (with one exception) the native insulin fold is retained in each analogue; the exception, PheB24----Gly, induces similar perturbations in both native insulin and DKP-insulin backgrounds. Notably, analogous substitutions exhibit parallel trends in receptor-binding potency over a wide range of affinities: D-PheB24 greater than unsubstituted greater than GlyB24 greater than SerB24 greater than AlaB25 greater than LeuB25 greater than SerB25, whether the substitution was in a native human or DKP-insulin background. Such "template independence" reflects an absence of functional interactions between the B24 and B25 sites and additional substitutions in DKP-insulin and demonstrates that mutations in discrete surfaces of insulin have independent effects on protein structure and function. In particular, the respective receptor-recognition (PheB24, PheB25), hexamer-forming (HisB10), and dimer-forming (ProB28, LysB29) surfaces of insulin may be regarded as independent targets for protein design. DKP-insulin provides an appropriate biophysical model for defining structure-function relationships in a monomeric template.

Amino Acid Sequence↗
PubMed1992

Structural biology and diabetes mellitus: molecular pathogenesis and rational drug design.

Emerging concepts in the aetiology and pathogenesis of Type 1 (insulin-dependent) diabetes mellitus may offer new opportunities for treatment and cure. Here we describe recent advances in structural molecular biology and molecular design relevant to rational drug discovery. Such approaches focus on the three-dimensional structures of macromolecules and their interactions. In the coming decade such techniques may be applied to a wide variety of diabetes-related targets.

Amino Acid Sequence↗
PubMed1992

The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals.

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.

Amino Acid Sequence↗
PubMed1992

Physicians' negative perception of sclerotherapy for venous disorders: review of a 7-year experience with modern sclerotherapy.

Sclerotherapy is the injection of a sclerosing substance directly into a varicosity or telangiectasia to cause damage, fibrosis, and eventual obliteration of the vessel. Although sclerotherapy has been shown to be effective with a low incidence of side effects, many physicians in the United States are unaware of the technique or view it in a negative way. To better define the extent of physician unfamiliarity or prejudice toward sclerotherapy, we surveyed a local medical community of gynecologists by anonymous written questionnaire. The results indicated that 82% of the responding physicians believed that their knowledge of sclerotherapy was insufficient to advise their patients, leaving patients to learn about sclerotherapy from mass media sources. Sclerotherapy was perceived as only slightly effective therapy for varicose veins and was thought to have a relatively high incidence of side effects. With recent improvements in the technique, our collaborative experience with 3000 patients receiving 50,000 injections over the past 5 years has shown that sclerotherapy is a safe procedure achieving outstanding cosmetic results with concomitant symptomatic relief of painful varicosities and telangiectases. Sclerotherapy, as practiced today, offers an effective, simple, and less costly alternative to surgical stripping for most varicose and telangiectatic veins. The survey data demonstrate the need to educate the American medical community about the technique and merits of modern sclerotherapy so that patients may be properly guided by their physicians to the best sources of care for their aching and unsightly leg veins.

Adolescent↗
PubMed1992

Structure of a receptor-binding fragment from human luteinizing hormone beta-subunit determined by [1H]- and [15N]nuclear magnetic resonance spectroscopy.

The structure of the glycoprotein hormones (LH, CG, FSH, and TSH) and their mechanism of receptor recognition are problems of long-standing interest and speculation. Here we describe the two-dimensional [1H]nuclear magnetic resonance ([1H]NMR) analysis of a linear peptide model for the intercysteine sequence (38-57) from the beta-subunit of human (h) LH. This sequence contains functional determinants for receptor binding and postreceptor activation and is predicted by computer-based modeling to fold as a compact minidomain containing a central amphipathic helix. To test this prediction, an Arg-extended disulfide-free (38-57) analog of enhanced solubility was prepared for complementary circular-dichroic and two-dimensional NMR studies. The linear peptide retains ovarian membrane receptor-binding activity. Although the peptide is not highly structured in aqueous solution, circular-dichroic analysis shows partial alpha-helix formation in a lipophilic medium (50% trifluoroethanol). Complete sequential assignment is obtained in 50% trifluoroethanol based on homonuclear and [15N]edited heteronuclear NMR methods. alpha-Helix-related (i,i + 3) connectivities are observed by nuclear-Overhauser effect spectroscopy that define an amphipathic alpha-helical segment (residues 41-48). Additional long range nuclear-Overhauser effects are observed in the C-terminal region that are consistent with beta-turns involving one or more proline residues; these may serve to reverse the direction of the peptide chain. A nuclear-Overhauser effect contact is identified between residues 38 and 55 at opposite ends of the linear sequence, suggesting that a loop configuration is significantly populated in this solvent system. These results, taken together, characterize elements of ordered structure in the 38-57 peptide, which appear to be distinguishing features of hLH (and the homologous region of hCG). We propose that the structure of this peptide provides a model for the structure of the corresponding region of native hLH in the hormone-receptor complex.

Amino Acid Sequence↗
PubMed1991

Receptor binding redefined by a structural switch in a mutant human insulin.

Crystal structures of insulin have been determined in various distinct forms, the relevance of which to receptor recognition has long been the subject of speculation. Recently the crystal structure of an inactive insulin analogue has been determined and, surprisingly, found to have a conformation identical to native insulin. On this basis Dodson and colleagues have suggested that the known insulin crystal structures reflect an inactive conformation, and that a change in conformation is required for activity--specifically, the carboxy terminal residues of the B-chain are proposed to separate from the amino terminal residues of the A-chain. Here we report the solution structure of an active insulin mutant, determined by two-dimensional NMR, which supports this hypothesis. In the mutant, the carboxy terminal beta-turn and beta-strand of the B-chain are destabilized and do not pack across the rest of the molecule. We suggest that analogous detachment of the carboxy terminal region of the B-chain occurs in native insulin on binding to its receptor. Our finding that partial unfolding of the B-chain exposes an alternative protein surface rationalizes the receptor-binding properties of a series of anomalous insulin analogues, including a mutant insulin associated with diabetes mellitus in man.

Amino Acid Sequence↗
PubMed1991

Alternating zinc fingers in the human male-associated protein ZFY: HX3H and HX4H motifs encode a local structural switch.

The two-finger repeat in the human male-associated protein ZFY provides a model for comparative 2D-NMR studies of classical and variant Zn fingers. This repeat is defined in part by an alternation in spacing between consensus (HX3H) and variant (HX4H) histidine spacings. To investigate the effects of a "switch" between alternative histidine spacings, we have designed an HX3H analogue of a representative HX4H domain of known structure [ZFY-6; Kochoyan, M., Havel, T., Nguyen, D. T., Dahl, C. E., Keutmann, H. T., & Weiss, M. A. (1991) Biochemistry 30, 3371-3386]. The HX3H analogue (designated ZFY-switch) forms a tetrahedral Co2+ complex whose thermodynamic stability is similar to that of the parent peptide. 2D-NMR studies demonstrate that ZFY-switch and ZFY-6, although similar in overall structure, exhibit significant local changes near the site of deletion. Whereas the HX4H site in the native finger forms a nonstandard loop, the HX3H site in ZFY-switch folds as a 3(10) extension of the C-terminal alpha-helix, as observed in the NMR solution structure of a consensus HX3H domain [Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A., & Wright, P. E. (1989) Science 245, 635-637] and in the crystal structure of a representative Zn finger-DNA complex [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. We propose that variant histidine spacings (HX3H and HX4H) encode a local switch between alternative surface architectures with implications for models of protein-DNA recognition.

Amino Acid Sequence↗
PubMed1991

Architectural rules of the zinc-finger motif: comparative two-dimensional NMR studies of native and "aromatic-swap" domains define a "weakly polar switch".

The Zn-finger motif, encoding a globular minidomain with characteristic structure, provides a striking example of a sequence template for protein folding. Insight into architectural rules relating the amino acid sequence of a protein to its structure and stability may be obtained by comparative study of analogues. As our first step toward defining such rules for the Zn finger, we have recently described the design of an "aromatic-swap" analogue based on the ZFY two-finger repeat: a conserved alternation in sequence pattern observed among odd- and even-numbered domains in a family of sex-related vertebrate transcription factors. Consensus and "swapped" aromatic residues, introduced as revertants of less stable "aromaticless" analogues, were observed to provide equivalent contributions to the thermodynamic stability of the Zn finger. Here we describe and compare the solution structures of a wild-type domain and an aromatic-swap analogue, as determined by two-dimensional NMR and distance-geometry/restrained molecular dynamics calculations. The wild-type and aromatic-swap analogue each contain an N-terminal beta-sheet and a C-terminal alpha-helix (beta beta alpha motif), as observed in other systems, and exhibit a highly ordered hydrophobic core in which the native or swapped aromatic ring is closely packed. Remarkably, however, the two structures are stabilized by alternative aromatic-aromatic interactions, which in turn alter the respective DNA-binding surfaces. Our results suggest that native and swapped Zn-finger sequences encode a "weakly polar switch" between thermodynamically equivalent but functionally distinct architectures for DNA recognition.

Amino Acid Sequence↗