Biomedical subjects

Hans-Georg Koch

Publications and source records attributed to Hans-Georg Koch.

At least 19 recordsLinked to original sources

PubMed2025

The small bacterial membrane protein YohP induces nucleoid condensation in E. coli and inhibits oligomerization of antimicrobial peptides.

Prokaryotic organisms execute multiple stress response mechanisms in order to cope with rapidly changing environments. Some mechanisms respond to specific cues, such as the OxyR-dependent response to hydrogen peroxide or the SOS-response that is induced upon DNA-damage. These specific responses complement general mechanisms that respond to multiple and diverse stressors. One example is nucleoid condensation, which is a rapid and effective mechanism for genome protection and observed in response to various stresses, including entry into stationary phase. Recently, the upregulation of small membrane proteins (SMPs) in response to stress was observed, but details on how this emerging class of proteins modulate the stress response is largely unknown. Here, we demonstrate that the production of two SMPs, YohP and YncL, cause nucleoid condensation in Escherichia coli. Nucleoid condensation is the result of YohP-/YncL-induced sublethal membrane depolarization, which induces the phage-shock response and leads to a reduction of global protein synthesis. YohP production also prevents the oligomerization of the antimicrobial peptide magainin-2 in the E. coli membrane and reduces the metabolic activity of E. coli cells. Thus, the synthesis of YohP and likely of other SMPs potentially protects bacterial cells against some unfavorable conditions by shifting them into a metabolically silent state.

YncL↗

PubMed2006

Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites.

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY-SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.

Bacterial Proteins↗

PubMed2006

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.

Adenosine Triphosphate↗

PubMed2006

Newborn screening for 3-methylcrotonyl-CoA carboxylase deficiency: population heterogeneity of MCCA and MCCB mutations and impact on risk assessment.

New technology enables expansion of newborn screening (NBS) of inborn errors aimed to prevent adverse outcome. In conditions with a large share of asymptomatic phenotypes, the potential harm created by NBS must carefully be weighed against benefit. Policies vary throughout the United States, Australia, and Europe due to limited data on outcome and treatability of candidate screening conditions. We elaborated the rationale for decision making in 3-methylcrotonyl-coenzyme A (CoA) carboxylase deficiency (MCCD), which afflicts leucine catabolism, with reported outcomes ranging from asymptomatic to death. In Bavaria, we screened 677,852 neonates for 25 conditions, including MCCD, based on elevated concentrations of 3-hydroxyisovalerylcarnitine (3-HIVA-C). Genotypes of MCCA (MCCC1) and MCCB (MCCC2) were assessed in identified newborns, their relatives, and in individuals (n = 17) from other regions, and correlated to biochemical and clinical phenotypes. NBS revealed eight newborns and six relatives with MCCD, suggesting a higher frequency than previously assumed (1:84,700). We found a strikingly heterogeneous spectrum of 22 novel and eight reported mutations. Allelic variants were neither related to biochemical nor anamnestic data of our probands showing all asymptomatic or benign phenotypes. Comparative analysis of case reports with NBS data implied that only few individuals (< 10%) develop symptoms. In addition, none of the symptoms reported so far can clearly be attributed to MCCD. MCCD is a genetic condition with low clinical expressivity and penetrance. It largely represents as nondisease. So far, there are no genetic or biochemical markers that would identify the few individuals potentially at risk for harmful clinical expression. The low ratio of benefit to harm was pivotal to the decision to exclude MCCD from NBS in Germany. MCCD may be regarded as exemplary of the ongoing controversy arising from the inclusion of potentially asymptomatic conditions, which generates a psychological burden for afflicted families and a financial burden for health care systems.

Alleles↗

PubMed2005

Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex.

The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.

Bacterial Proteins↗

PubMed2005

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli.

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.

Adenosine Triphosphatases↗

PubMed2005

FtsY, the bacterial signal-recognition particle receptor, interacts functionally and physically with the SecYEG translocon.

Co-translational membrane targeting of proteins by the bacterial signal-recognition particle (SRP) requires the specific interaction of the SRP-ribosome nascent chain complex with FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha-subunit of the eukaryotic SR, which is tethered to the endoplasmic-reticulum membrane by its interaction with the integral SRbeta-subunit. In contrast to SRalpha, FtsY is partly membrane associated and partly located in the cytosol. However, the mechanisms by which FtsY associates with the membrane are unclear. No gene encoding an SRbeta homologue has been found in bacterial genomes, and the presence of an FtsY-specific membrane receptor has not been shown so far. We now provide evidence for the direct interaction between FtsY and the SecY translocon. This interaction offers an explanation of how the bacterial SRP cycle is regulated in response to available translocation channels.

Bacterial Proteins↗

PubMed2004

A common mutation is associated with a mild, potentially asymptomatic phenotype in patients with isovaleric acidemia diagnosed by newborn screening.

Isovaleric acidemia (IVA) is an inborn error of leucine metabolism that can cause significant morbidity and mortality. Since the implementation, in many states and countries, of newborn screening (NBS) by tandem mass spectrometry, IVA can now be diagnosed presymptomatically. Molecular genetic analysis of the IVD gene for 19 subjects whose condition was detected through NBS led to the identification of one recurring mutation, 932C-->T (A282V), in 47% of mutant alleles. Surprisingly, family studies identified six healthy older siblings with identical genotype and biochemical evidence of IVA. Our findings indicate the frequent occurrence of a novel mild and potentially asymptomatic phenotype of IVA. This has significant consequences for patient management and counseling.

Algorithms↗

PubMed2004

An activated 5' cryptic splice site in the human ALG3 gene generates a premature termination codon insensitive to nonsense-mediated mRNA decay in a new case of congenital disorder of glycosylation type Id (CDG-Id).

A defect of the dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl mannosyltransferase encoded by the ALG3 gene (alias NOT56L) causes congenital disorder of glycosylation type Id (CDG-Id). In this work, a new mutation in the ALG3 gene causing atypical splicing is described with characterization of expression levels and transcript stabilities of the different splice products. A silent mutation in exon 1 of the ALG3 gene (c.165C<T) resulted in a deletion in the corresponding transcripts (c.160_196del) due to the activation of a cryptic donor splice site. Expression studies revealed that negligible amounts of normal transcripts were present in the patient. The deletion in the ALG3 gene generated a premature termination codon (PTC) coding for an ALG3 protein truncated after the first N-terminal transmembranous domain (p.Val54fsX66). Nonsense mediated decay (NMD) of mRNA is a general mechanism for clearing of RNA molecules containing suitable PTCs. However, suppression of NMD using cycloheximide had no influence on ALG3 transcript levels, although the PTCs of the transcript fulfill the criteria for the initiation of NMD. The results presented in this work demonstrate that factors abrogating NMD of the ALG3 gene exists and that the ALG3 gene can serve as a valuable tool for further investigations of the regulation of NMD.

Amino Acid Sequence↗

PubMed2004

Successful hematopoietic stem cell transplantation in Farber disease.

Farber disease, a lysosomal storage disorder, has a dismal prognosis leading to death with progressive granulomatous inflammation, even in patients without central nervous system involvement (type 2/3). We report the first successful hematopoietic stem cell transplantations in 2 patients with Farber disease type 2/3, resulting in resolution of symptoms.

Child, Preschool↗

PubMed2004

The increased lysis of fetal cells in the mother after pregnancies complicated by pre-eclampsia or HELLP syndrome is not the result of a specific anti-fetal cytotoxicity of the mother.

PROBLEM: In pregnancies complicated by pre-eclampsia or hemolytisis elevated liver enzymes, low platelets (HELLP) syndrome, an increase of fetal DNA in the maternal serum indicates an increased lysis of fetal cells. Whether this cytolysis is the cause or the result of an increased specific cytotoxicity against the fetal cells is not yet known. METHODS: Ten mothers after healthy pregnancy, eight mothers after pregnancy complicated by prematurity (GA < 37 weeks), and eight mothers with pregnancies complicated by pre-eclampsia or HELLP syndrome and their male children were enrolled in the study. Fetally derived DNA in the maternal serum and the specific anti-fetal cytotoxicity of the maternal lymphocytes were measured. RESULTS: Detection of fetal DNA in maternal serum was significantly associated with pre-eclampsia/HELLP syndrome but not with anti-fetal cytotoxicity of the maternal lymphocytes. CONCLUSION: The cytolysis of fetal cells in mothers after pregnancies complicated by pre-eclampsia/HELLP syndrome is neither the reason for nor the result of an increased specific anti-fetal cytotoxicity.

Adult↗

PubMed2004

Enzyme replacement therapy administered during hemodialysis in patients with Fabry disease.

BACKGROUND: Enzyme replacement treatment with recombinant human alpha-galactosidase A (r-halphaGalA) is now available for patients with Fabry disease, many of whom are on maintenance hemodialysis. Because r-halphaGalA must be infused over several hours, administering the enzyme during dialysis would save a day of treatment for patients receiving both therapies. However, these procedures have never been combined due to concerns about possible loss of enzyme in the dialysate. METHODS: Ten Fabry patients received r-halphaGalA (1 mg/kg body weight continuously infused over 4 hours) during dialysis and separately in the interval between dialysis treatments. Plasma activity of r-halphaGalA was measured at baseline and then every hour for both procedures. In two patients, a third r-halphaGalA infusion during dialysis with a high-flux membrane was followed. RESULTS: The rise in plasma concentrations of r-halphaGalA during infusion and the steady-state levels reached were comparable for enzyme administrations with or without dialysis. The trend for the somewhat higher activities during hemodialysis was explained by volume contraction due to ultrafiltration. With the use of a high-flux dialyzer, the plasma r-halphaGalA activities were identical to those that were observed during low-flux dialysis. CONCLUSIONS: Administration of r-halphaGalA during hemodialysis is not associated with a reduced activity of r-halphaGalA therapy in patients with Fabry disease. Replacement therapy with r-halphaGalA may therefore be performed during hemodialysis without apparent loss of enzyme into the dialysate.

Fabry Disease↗

PubMed2004

Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide.

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.

Adenosine Triphosphatases↗

PubMed2003

Ligand crowding at a nascent signal sequence.

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.

Bacterial Outer Membrane Proteins↗

PubMed2003

Neonatal screening for citrullinaemia.

UNLABELLED: In a period of 40 months (1st March 1999 to 30th June 2002) 610,000 blood samples were analysed in one screening centre for citrulline as a pilot study for neonatal screening using tandem mass spectrometry. Persistent hypercitrullinaemia (Cit >1.5 mg/dl or 85.5 micro mol/l, not corrected for recovery) was identified in 15 newborns. Four children were diagnosed with classical neonatal onset citrullinaemia and eight with persisting asymptomatic hypercitrullinaemia. In two asymptomatic newborns and in one symptomatic preterm patient, argininosuccinate lyase deficiency was identified as the cause of moderately elevated levels of citrulline (cases not described in this paper). Citrulline concentrations were only temporarily mildly elevated in two newborns and in these the results of the original neonatal screening were therefore regarded as false-positive; we did not find any other false-positives. The screening result allowed the introduction of immediate specific treatment in two cases of citrullinaemia and may have prevented metabolic decompensation in those with presumed mild citrullinaemia. In one child who developed severe hyperammonaemia on the 2nd day of life, sequelae could not be avoided. CONCLUSION: neonatal screening for citrullinaemia is more complex than expected and, with the actual logistics, results may be obtained too late in severe forms.

Argininosuccinic Aciduria↗

PubMed2003

Export of beta-lactamase is independent of the signal recognition particle.

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.

Biological Transport↗

PubMed2003

Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.

Cell Membrane↗

PubMed2002

The integration of YidC into the cytoplasmic membrane of Escherichia coli requires the signal recognition particle, SecA and SecYEG.

The integration of the polytopic membrane protein YidC into the inner membrane of Escherichia coli was analyzed employing an in vitro system. Upon integration of in vitro synthesized YidC, a 42-kDa membrane protected fragment was detected, which could be immunoprecipitated with polyclonal anti-YidC antibodies. The occurrence of this fragment is in agreement with the predicted topology of YidC and probably encompasses the first two transmembrane domains and the connecting 320-amino acid-long periplasmic loop. The integration of YidC was strictly dependent on the signal recognition particle and SecA. YidC could not be integrated in the absence of SecY, SecE, or SecG, suggesting that YidC, in contrast to its mitochondrial orthologue Oxa1p, cannot engage a SecYEG-independent protein-conducting channel.

Adenosine Triphosphatases↗