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Biomedical subjects

G Schatz

Publications and source records attributed to G Schatz.

At least 37 records · Page 2Linked to original sources
PubMed1998

Immunogenicity of hepatitis B Vaccines. Implications for persons at occupational risk of hepatitis B virus infection.

OBJECTIVE: To assess risk factors for decreased immunogenicity among adults vaccinated with hepatitis B vaccine and to determine the importance of differences in immunogenicity between vaccines among health care workers (HCWs). DESIGN: Randomized clinical trial and decision analysis. PARTICIPANTS: HCSw. MAIN OUTCOME MEASURES: Development of seroprotective levels of antibody to hepatitis B surface antigen (anti-HBs) and the number of expected chronic hepatitis B virus (HBV) infections associated with lack of protection. RESULTS: Overall, 88% of HCWs developed seroprotection. Risk factors associated with failure to develop seroprotection included increasing age, obesity, smoking and male gender (P < .05). Presence of a chronic disease was associated with lack of seroprotection only among persons > or = 40 years of age (P < .05). The two vaccines studied differed in their overall seroprotection rates (90% vs. 86%; P < .05), however, this difference was restricted to persons > or = 40 years of age (87% vs. 81%; P < .01). Among HCWs > or = 40 years of age, the decision analysis found 44 (0.34/100,000 person-years) excess chronic HBV infections over the working life of the cohort associated with use of the less immunogenic vaccine compared to the other. CONCLUSIONS: He patitis B vaccines are highly immunogenic, but have decreased immunogenicity associated with increasing age, obesity, smoking, and male gender; and among older adults, the presence of a chronic disease. One of the two available vaccines is more immunogenic among older adults; however, this finding has little clinical or public health importance. Hepatitis B vaccines should be administered to persons at occupational risk for HBV infection early in their career, preferably while they are still in their training.

Adult↗
PubMed1997

Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.

Bacterial Proteins↗
PubMed1997

The mitochondrial hsp70 chaperone system. Effect of adenine nucleotides, peptide substrate, and mGrpE on the oligomeric state of mhsp70.

Mitochondrial hsp70 (mhsp70) is a key component in the import and folding of mitochondrial proteins. In both processes, mhsp70 cooperates with the mitochondrial nucleotide exchange factor mGrpE (also termed Mge1p). In this work we have characterized the self-association of purified mhsp70, the interaction of mhsp70 with isolated mGrpE and protein substrate, and the effect of nucleotides on these interactions. mhsp70 can form oligomers that are dissociated by ATP or by a nonhydrolyzable ATP analog. A substrate peptide binds to mhsp70 in the absence of added nucleotides and is released by ATP but not by ADP. Binding of the peptide causes nucleotide-independent dissociation of the mhsp70 oligomers and enhances the mhsp70 ATPase. Purified mGrpE forms a homodimer. In the absence of added nucleotides, one mGrpE dimer binds to one molecule of mhsp70, forming a stable 122 kDa hetero-oligomer. This complex is weakened by ADP and completely dissociated by ATP.

Adenosine Diphosphate↗
PubMed1997

Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.

We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex.

Biological Transport, Active↗
PubMed1997

Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones.

We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import-stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor-MSF-Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor-Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor-MSF-Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20-precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.

14-3-3 Proteins↗
PubMed1997

Sequential action of two hsp70 complexes during protein import into mitochondria.

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Adenosine Diphosphate↗
PubMed1997

What is the driving force for protein import into mitochondria?

Nuclear-encoded mitochondrial proteins are synthesized in the cytosol as precursors and then imported into mitochondria. Protein import into the matrix space requires the function of the mitochondrial hsp70 (mhsp70) chaperone. mhsp70 is an ATPase that acts in conjunction with two partner proteins: the Tim44 subunit of the inner membrane import complex, and the nucleotide exchange factor mGrpE. A central question concerns how mhsp70 uses the energy of ATP hydrolysis to transport precursor proteins into the matrix. Recent evidence suggests that mhsp70 is a mechanochemical enzyme that actively pulls precursors across the inner membrane.

Adenosine Triphosphate↗
PubMed1997

ATP-dependent proteases that also chaperone protein biogenesis.

The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may function in the quality control of protein biogenesis.

ATP-Dependent Proteases↗
PubMed1997

Analysis of cleavage site mutations between the NC and PR Gag domains of Rous sarcoma virus.

In retroviruses, the viral protease (PR) is released as a mature protein by cleavage of Gag, Gag-Pro, or Gag-Pro-Pol precursor polypeptides. In avian sarcoma and leukemia viruses (ASLV), PR forms the C-terminal domain of Gag. Based on the properties of a mutation (cs22) in the cleavage site between the upstream NC domain and the PR domain, the proteolytic liberation of PR previously was inferred to be essential for processing of Gag and Pol proteins. To study this process in more detail, we have analyzed the effects that several mutations at the NC-PR cleavage site have on proteolytic processing in virus-like particles expressed in COS and quail cells. Mutant Gag proteins carrying the same mutations also were synthesized in vitro and tested for processing with purified PR. In both types of studies, N-terminal sequencing of the liberated PR domain was carried out to exactly identify the site of cleavage. Finally, synthetic peptides corresponding to the mutant proteins were assessed for the ability to act as substrates for PR. The results were all consistent and led to the following conclusions. (i) In vivo, if normal processing between NC and PR is prevented by mutations, limited cleavage occurs at a previously unrecognized alternative site three amino acids downstream, i.e., in PR. This N-terminally truncated PR is inactive as an enzyme, as inferred from the global processing defect in cs22 and a similar mutant. (ii) In Gag proteins translated in vitro, purified PR cleaves this alternative site as rapidly as it does the wild-type site. (iii) Contrary to previously accepted rules describing retroviral cleavage sites, an isoleucine residue placed at the P1 position of the NC-PR cleavage site does not hinder normal processing. (iv) A proline residue placed at the P2 position in this cleavage site blocks normal processing.

Animals↗
PubMed1997

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria.

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.

Adenosine Triphosphate↗
PubMed1996

Translocation arrest of an intramitochondrial sorting signal next to Tim11 at the inner-membrane import site.

The import of proteins from the cytosol into the mitochondrial matrix involves the concerted action of two separate import systems: the TOM system in the outer membrane, and the TIM system in the inner membrane. Here we report that the inner-membrane system also sorts proteins to the intermembrane space. Some intermembrane-space proteins, such as cytochromes b2 and c1, are synthesized with a complex pre-sequence consisting of a positively charged matrix targeting signal followed by an uncharged sequence that acts as sorting signal for the intermembrane space. We show that this sorting signal can be efficiently crosslinked to an inner-membrane protein of relative molecular mass 11K after the mature part of the precursor has been sorted to the intermembrane space. The 11K protein, which we term Tim11, is a component of the protein import system in the inner membrane.

Adenosine Triphosphate↗
PubMed1996

Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon.

Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein assembly, but not in degradation, were suppressed by overproduction of yeast mitochondrial Lon, an ATP-dependent serine protease. Suppression by Lon was enhanced by inactivation of the proteolytic site and was prevented by mutation of the ATP-binding site. It is suggested that the mitochondrial proteases Lon, Afg3p, and Rca1p can also serve a chaperone-like function in the assembly of mitochondrial protein complexes.

ATP-Dependent Proteases↗
PubMed1996

Common principles of protein translocation across membranes.

Most major systems that transport proteins across a membrane share the following features: an amino-terminal transient signal sequence on the transported protein, a targeting system on the cis side of the membrane, a hetero-oligomeric transmembrane channel that is gated both across and within the plane of the membrane, a peripherally attached protein translocation motor that is powered by the hydrolysis of nucleoside triphosphate, and a protein folding system on the trans side of the membrane. These transport systems are divided into two families: export systems that export proteins out of the cytosol, and import systems that transport proteins into cytosol-like compartments.

Amino Acid Sequence↗