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G Semenza

Publications and source records attributed to G Semenza.

At least 19 recordsLinked to original sources
PubMed2001

Imaging the native structure of the chaperone protein GroEL without fixation using atomic force microscopy.

Most sample preparation methods for scanning probe or electron microscopy require that biomolecules, such as proteins, be fixed. Fixation destroys the molecular functionality and can possibly affect the true molecular structure. Here we report sample preparation conditions that allow the imaging of an unfixed protein, GroEL, under in-vivo conditions, by atomic force microscopy. Under these conditions, the protein should maintain its native structure and biological activity. The typical toroidal shape with pore of the GroEL complex was easily visible in the images. Images of a single complex show dimensions that agree well with crystallographic data. Under in-vivo conditions, it should be possible to study the biological activity and function of proteins.

Chaperonin 60↗
PubMed1999

Species differences in the sites of cleavage of pro-lactase to lactase supports lack of selective pressure.

The pro-sequences in pro-lactase-phlorizin hydrolase (LPH) are needed for lactase to proceed past the ER, but are irrelevant as to the enzymatic activities. Hence, in all species removal of the pro- sequences (or most of them) must take place after the ER. Contrary to this, the details of the removal of these pro-sequences are to be expected to differ in the various species, since they are not subjected to selective pressure. Using site-directed mutagenesis we investigated processing in rabbit. The first cleavage occurs by furin (or furin-like PCs) and takes place at R-A-A-R(349) in the pro-sequence, generating the known 180 kDa intermediate. Replacing R(349) by Q results in a mutant which is not cleaved but nevertheless transported to the cell surface as demonstrated by immunofluorescence. Further processing of either the 180 kDa intermediate or the mutant is not directly mediated by furin-like PCs, but involves (also) other proteases. These results demonstrate that formation of the 180 kDa intermediate, consistently found only in rabbits, but not in man, is not essential for lactase transport: in all likelihood lack of selective pressure has led to species-specific processing of pro-LPH.

Animals↗
PubMed1998

Processing of human intestinal prolactase to an intermediate form by furin or by a furin-like proprotein convertase.

Human lactase-phlorizin hydrolase (human-LPH) is synthesized as a large precursor (prepro-LPH), then cleaved to a pro-LPH of 220 kDa which is further cut to a "mature-like LPH" of a size close to that of mature LPH, i.e. about 150 kDa (in the processing of rabbit pro-LPH the intermediate has a mass of approximately 180 kDa). By coexpression of human prepro-LPH with furin in COS-7 cells we show that furin generates a mature-like LPH. Radioactive amino acid sequence analysis reveals that furin recognizes the motif R-T-P-R832, a protein convertase consensus, to generate a NH2 terminus located 36 amino acids upstream of the NH2 terminal found in vivo at Ala869. This intermediate is ultimately cleaved to the mature LPH form by other proteases including the pancreatic ones. These data demonstrate that human pro-LPH, like the rabbit enzyme, is processed to the mature enzyme by furin or furin-like enzymes through at least an intermediate form that has, however, an apparent mass close to that of the mature enzyme.

Amino Acid Sequence↗
PubMed1998

Preparation of basal cell membranes for scanning probe microscopy.

Scanning probe microscopy has the potential for investigating membranes in a physiological environment. We prepared with a lysis-squirting protocol basal cell membranes, that are suitable for scanning probe microscopy. Investigations using atomic force microscopy under liquid revealed cellular filaments which correlated perfectly with fluorescently stained actin filaments. Globular structures with a diameter as little as 10 nm could be resolved by stripping cytoplasmic components from the membranes. Therefore, cytoplasmic sides of supported basal cell membranes prove useful to gain high resolution with scanning probe microscopy in studies of plasma membrane associated structures and processes under buffer solution.

Animals↗
PubMed1998

Intestinal lactase-phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro-LPH maturation.

Brush border lactase-phlorizin hydrolase carries two catalytic sites. In the human enzyme lactase comprises Glu-1749, phlorizin hydrolase Glu-1273. The proteolytic processing of pro-lactase-phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N-terminus of 'mature' final, brush border lactase-phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.

Amino Acid Sequence↗
PubMed1998

Measuring elasticity of biological materials by atomic force microscopy.

Atomic force microscopy (AFM) has proved its value not only for resolving the topographical structure of biological samples, but also for probing inherent properties of biological structures, like local interaction forces, mechanical properties or dynamics in a natural (physiological) environment. This minireview focuses on the acquisition of elasticity data of biomaterials by AFM. A possible theoretical model is presented, followed by a practical 'how to do it with AFM', and, finally, a brief overview of publications in this field is given.

Animals↗
PubMed1998

Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.

Adenosine Triphosphate↗
PubMed1997

"Adult only" esterase/phospholipase A of the small-intestine brush border membrane: isolation, identification of the catalytic site, and biosynthesis.

We have isolated and investigated an esterase/phospholipase A (EC 3.1.1) which is an intrinsic protein of the small-intestine brush border membrane of adult but not of preweaning rabbits. This enzyme had been referred to previously as AdRabB [Boll, W., Schmid-Chanda, T., Semenza, G., & Mantei, N. (1993) J. Biol. Chem. 268, 12901-12911]. Its amino acid sequence shows four extensive, homologous repeats between the signal sequence and a hydrophobic stretch in the C-terminal region. We have identified a serine residue (Ser400) in the (unexpectedly) single catalytic site and indicate that this esterase is likely to operate by way of a Ser-His-Asp/Glu triad. This esterase/phospholipase A is synthesized as a single polypeptide chain of 170-180 kDa, agreeing well with the size deduced from its cognate cDNA sequence [Boll, W., et al. (1993) J. Biol. Chem. 268, 12901-12911], and it undergoes (at least) N-glycosylation. Once it has reached the brush border membrane, it is subjected in vivo to two types of proteolytic processing, presumably by pancreatic protease(s): a split after Arg263, with loss of the first N-terminal repeat, and two or more cleavages much farther downstream but still located in the luminal bulk of the protein; these splits lead to multiple bands of approximately 140, 125, 100, and 90 kDa.

Amino Acid Sequence↗
PubMed1997

Lactase persistence versus decline in human adults: multifactorial events are involved in down-regulation after weaning.

BACKGROUND & AIMS: In nonhuman mammals, lactase activity declines during or after weaning. In contrast, about one half of the human species maintains high lactase activity even in adulthood. To clarify this difference, this study examined some parameters for which contrasting observations have been reported in connection with lactase decline. METHODS: Lactase activity, lactase messenger RNA (mRNA) levels, and in vitro lactase biosynthesis were determined in normal jejunal samples from a large group of white adults, all born in or near Naples. RESULTS: Of 44 individuals, 10 were lactase persistent and 34 were hypolactasic. Biosynthesis of prolactase correlated well with lactase mRNA levels, indicating transcriptional control; it did less so with steady-state lactase activity. Examination of lactase mRNA levels and lactase activity/lactase mRNA ratios revealed a heterogeneous pattern of lactase mRNA level, lactase synthesis, and activity in both lactase persistent and hypolactasic subjects. CONCLUSIONS: Both transcriptional and posttranscriptional factors cause the decline of intestinal lactase. This probably explains the multifarious observations that most studies on adult-type hypolactasia have reported. The single overriding factor distinguishing lactase-persistent subjects from hypolactasic subjects is the high rate of lactase biosynthesis.

Adult↗
PubMed1996

Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.

The intestinal effects of Clostridium difficile toxin A are inidated by toxin binding to luminal enterocyte receptors. We reported previously that the rabbit ileal brush border (BB) receptor is a glycoprotein with an alpha-d-galactose containing trisaccharide in the toxin-binding domain (1991. J. Clin. Invest. 88:119-125). In this study we characterized the rabbit ileal BB receptor for this toxin. Purified toxin receptor peptides of 19 and 24 amino acids showed 100% homology with rabbit sucrase-isomaltase (SI). Guinea pig receptor antiserum reacted in Western blots with rabbit SI and with the purified toxin receptor. Antireceptor IgG blocked in vitro binding of toxin A to rabbit ileal villus cell BB. Furthermore, anti-SI IgG inhibited toxin A-induced secretion (by 78.1%, P < 0.01), intestinal permeability (by 80.8%, P < 0.01), and histologic injury (P < 0.01) in rabbit ileal loops in vivo. Chinese hamster ovary cells transfected with SI cDNA showed increased intracellular calcium increase in response to native toxin (holotoxin) or to a recombinant 873-amino acid peptide representing the receptor binding domain of toxin A. These data suggest that toxin A binds specifically to carbohydrate domains on rabbit ileal SI, and that such binding is relevant to signal transduction mechanisms that mediate in vitro and in vivo toxicity.

Amino Acid Sequence↗
PubMed1996

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.

Enzymes, Immobilized↗
PubMed1995

Phosphorylation of the N-terminal intracellular tail of sucrase-isomaltase by cAMP-dependent protein kinase.

This paper reports the phosphorylation of the intracellular N-terminal tail of sucrase-isomaltase by protein kinase A and shows that this phosphorylation is targeted to Ser6 within a sequence Arg/Lys/Lys-Phe-Ser, which is conserved in all sucrase-isomaltase sequences known so far. By dephosphorylation of native sucrase-isomaltase with an immobilized acid phosphatase and rephosphorylation with protein kinase A, it is shown that Ser6 may be partially phosphorylated in vivo, raising the possibility that the tail itself and its phosphorylation by protein kinase A may be physiologically significant.

Amino Acid Sequence↗
PubMed1995

Furin, PC1/3, and/or PC6A process rabbit, but not human, pro-lactase-phlorizin hydrolase to the 180-kDa intermediate.

Small intestinal lactase-phlorizin hydrolase (LPH) is synthesized as a large precursor (prepro-LPH) of 1926 amino acids. In the endoplasmic reticulum, prepro-LPH is split by signal protease. The resulting pro-LPH is cut to mature LPH directly (human) or via a 180-kDa intermediate (rabbit), most likely in the trans-Golgi network or in a later compartment. Antibodies directed against different regions of rabbit pro-LPH locate the cleavage site resulting in the 180-kDa intermediate between amino acid residues 79 and 286. This stretch contains the two sequences -Arg-Cys-Tyr-Arg114 approximately -Arg-Ala-Ser-Arg191 approximately, which are potential cleavage sites for subtilisin-like proprotein convertases. These sites are not conserved in human pro-LPH. By coexpression in COS 7 cells of rabbit prepro-LPH and proprotein convertases (PC 1/3, PC2, PC6A, PC6B, furin), we show that furin, PC 1/3, and PC6A generate a processing intermediate that is immunologically indistinguishable from the one observed in vivo. Furin, PC 1/3, and PC6A are all expressed in the small intestine as shown by a polymerase chain reaction-based approach and, more specifically, in enterocytes, as shown by in situ hybridization. These results suggest that furin, PC 1/3, and/or PC6A are responsible for the in vivo processing of rabbit pro-LPH to the 180-kDa intermediate.

Amino Acid Sequence↗
PubMed1995

Disposition of the carboxy-terminus tail of rabbit lactase-phlorizin hydrolase elucidated by phosphorylation with protein kinase A in vitro and in tissue culture.

The intracellular disposition of the carboxy-terminus tail of rabbit lactase-phlorizin hydrolase (LPH) is demonstrated, using a specific phosphorylation of Ser1916 by protein kinase A (PKA). This phosphorylation is shown to occur not only in vitro (with pure LPH and pure catalytic subunit of PKA), but also in an organ culture of the small intestine. Cholera toxin, which is known to act in vivo on the membranes of the small intestine, with severe clinical consequences, and to elevate the intracellular cyclic AMP of enterocytes, is shown to enhance significantly the phosphorylation of LPH in intact cells grown as an organ culture. These findings establish the cytosolic orientation of the carboxy-terminus tail of LPH in situ, and raise the possibility that the tail itself and its phosphorylation by PKA may have a physiological or physiopathological significance.

Amino Acid Sequence↗