Biomedical subjects

K Beck

Publications and source records attributed to K Beck.

At least 19 recordsLinked to original sources

PubMed2004

Rapid alleviation of signs and symptoms of rheumatoid arthritis with intravenous or subcutaneous administration of adalimumab in combination with methotrexate.

OBJECTIVE: This randomized, placebo-controlled, double-blind, Phase 1 study assessed the magnitude, onset, and duration of response with intravenous (i.v.) and subcutaneous (s.c.) adalimumab (Humira, Abbott Laboratories) combined with methotrexate (MTX) in patients with active rheumatoid arthritis (RA) despite previous MTX therapy. METHODS: Fifty-four patients were randomized to two injections of i.v. or s.c. adalimumab (1 mg/kg) or placebo while continuing on MTX (mean dose, 15.7 mg/week). Dosing intervals were determined by the European League Against Rheumatism (EULAR) response criteria, and were allowed to range from 1 to 3 months. Efficacy was mainly assessed using the EULAR response criteria and the American College of Rheumatology (ACR) response criteria. RESULTS: Moderate EULAR response was achieved at least once within 29 days after the first injection in 83% and 61% of patients receiving i.v. and s.c. adalimumab respectively, compared with 44% for placebo [probability (p) < or = 0.05 for i.v. adalimumab versus placebo]. A 20% improvement in disease activity according to the ACR criteria (ACR20 response) was achieved by 72% and 67% of patients receiving i.v. and s.c. adalimumab respectively, compared with 28% for placebo (p < or = 0.01 and p < or = 0.05, respectively, versus placebo). By Day 15 after the first and second injections, statistically significant moderate EULAR and ACR20 response rates were achieved with either i.v. or s.c. adalimumab compared with placebo (p < or = 0.05). The mean times to second injection for i.v. adalimumab, s.c. adalimumab, and placebo were 42.2 days (range: 27-84 days), 38.3 days (range: 26-85 days), and 28.4 days (range: 26-32 days), respectively (minimum time allowed by the protocol between the first and second injections was 4 weeks). Adalimumab in combination with MTX was well tolerated, with no patients being withdrawn because of adverse events. CONCLUSION: Either i.v. or s.c. adalimumab added to MTX significantly improved the signs and symptoms of RA compared with MTX alone. Subcutaneously administered adalimumab appeared to provide a response that was as great, as rapid, and as enduring as that with i.v. adalimumab.

Adalimumab↗

PubMed2003

Efficacy and safety of the fully human anti-tumour necrosis factor alpha monoclonal antibody adalimumab (D2E7) in DMARD refractory patients with rheumatoid arthritis: a 12 week, phase II study.

OBJECTIVES: To evaluate efficacy, dose response, safety, and tolerability of adalimumab (D2E7) in disease modifying antirheumatic drug (DMARD) refractory patients with longstanding, active rheumatoid arthritis (RA). METHODS: During a 12 week, double blind, placebo controlled study, 284 patients were randomly allocated to receive weekly subcutaneous injections of adalimumab 20 mg (n = 72), 40 mg (n = 70), or 80 mg (n = 72) or placebo (n = 70) without concomitant DMARDs. RESULTS: Adalimumab significantly improved the signs and symptoms of RA for all efficacy measures. ACR20 responses with adalimumab were significant at each assessment versus placebo (p</=0.01). Additionally, ACR responses with adalimumab were achieved more rapidly than with placebo, with 82/115 (71%) of the ultimate ACR20 response rate to adalimumab treatment achieved at week 2. At week 12, for adalimumab 20, 40, and 80 mg, ACR20 response rates were 50.7%, 57.1%, and 54.2%, respectively, versus 10.0% for placebo (p</=0.001 for all comparisons); ACR50 rates were 23.9%, 27.1%, and 19.4%, respectively, versus 1.4% for placebo (p</=0.001 for all comparisons); and ACR70 rates were 11.3%, 10.0%, and 8.3%, respectively, versus 0.0% for placebo (p</=0.05 for all comparisons). All adalimumab doses significantly improved all ACR core criteria at all assessments. The 40 mg and 80 mg doses provided similar benefit. Adalimumab at all doses was generally well tolerated, with only mild or moderate adverse events. Completion rates were 87% for adalimumab and 67% for placebo. CONCLUSIONS: Adalimumab given as monotreatment to patients with longstanding, severe RA refractory to traditional DMARDs produced a rapid, sustained response and was safe and well tolerated, with no dose limiting side effects.

Adalimumab↗

PubMed2002

Impact of sepsis, lung injury, and the role of lipid infusion on circulating prostacyclin and thromboxane A(2).

OBJECTIVE: To investigate whether plasma levels of prostacyclin (PGI2) and thromboxane A(2) (TxA2) are a function of the infusion rate of soybean-based fat emulsions, severity of systemic inflammation, and pulmonary organ failure. DESIGN: Prospective, randomized, crossover study. SETTING: Intensive care unit of a university hospital. PATIENTS: Eighteen critically ill patients, ten presenting with severe sepsis, eight with SIRS or sepsis complicated with ARDS. INTERVENTIONS: Patients were randomly assigned to receive rapid fat infusion over 6 h (rFI) or slow fat infusion over 24 h (sFI) along with parenteral nutrition. MEASUREMENTS AND RESULTS: The stable prostanoids 6-keto-PGF1alpha and TxB2 were measured in arterial and mixed venous blood samples, and at 6-h periods trans-pulmonary balances (TPB) were calculated. Free linoleic acid fraction was determined in arterial blood. rFI induced greater increase of linoleic acid than sFI in both groups. Enhanced prostanoid levels and correlations with linoleic acid availabilities were found, however, in ARDS patients only, revealing the highest sepsis- and lung injury scores. Averaged TPB per 24 h was positive in the sepsis group and negative in the ARDS group as rFI induced lowest TPB values for TxB2 at 6 h. CONCLUSION: The quantity of prostanoids formed and their subsequent utilization are dependent on the availability of precursor linoleic acid and are probably affected by the severity of SIRS or sepsis and the existence of pulmonary organ failure, respectively. Because TxA2 might be extracted by the injured lung, rapid infusion of soybean-based fat emulsions should be avoided in patients suffering from severe pulmonary organ failure.

Adult↗

PubMed2001

[Control of tachycardia with intravenous amiodarone in acute left heart failure].

The treatment of atrial tachycardia in critically ill patients can be difficult. Nine cases were presented with atrial tachyarrhythmias (mean heart rate > 130 beats/min) and left heart failure. Congestive heart failure was diagnosed in 6 patients (ejection fraction < 25%) and hypertensive heart failure in 3 patients (ejection infarction > 55%). The infusion of amiodarone (450 mg over 10 min and 0.5 mg/min after the bolus administration) was associated with a decrease in heart rate 31 beats/min and an increase in systolic blood pressure of 13 mm Hg after one hour. There was only one adverse effect secondary to amiodarone therapy. In this case the sinus rhythm converted within 24 hours but T-waves alternans and short running torsade de pointes ventricular tachycardia was observed and amiodarone therapy was discontinued.

Acute Disease↗

PubMed2001

A Drosophila haemocyte-specific protein, hemolectin, similar to human von Willebrand factor.

We identified a novel Drosophila protein of approximately 400 kDa, hemolectin (d-Hml), secreted from haemocyte-derived Kc167 cells. Its 11.7 kbp cDNA contains an open reading frame of 3843 amino acid residues, with conserved domains in von Willebrand factor (VWF), coagulation factor V/VIII and complement factors. The d-hml gene is located on the third chromosome (position 70C1-5) and consists of 26 exons. The major part of d-Hml consists of well-known motifs with the organization: CP1-EG1-CP2-EG2-CP3-VD1-VD2-VD'-VD3-VC1-VD"-VD"'-FC1-FC2-VC2-LA1-VD4-VD5-VC3-VB1-VB2-VC4-VC5-CK1 (CP, complement-control protein domain; EG, epidermal-growth-factor-like domain; VB, VC, VD, VWF type B-, C- and D-like domains; VD', VD", VD"', truncated C-terminal VDs; FC, coagulation factor V/VIII type C domain; LA, low-density-lipoprotein-receptor class A domain; CK, cysteine knot domain). The organization of VD1-VD2-VD'-VD3, essential for VWF to be processed by furin, to bind to coagulation factor VIII and to form interchain disulphide linkages, is conserved. The 400 kDa form of d-Hml was sensitive to acidic cleavage near the boundary between VD2 and VD', where the cleavage site of pro-VWF is located. Agarose-gel electrophoresis of metabolically radiolabelled d-Hml suggested that it is secreted from Kc167 cells mainly as dimers. Resembling VWF, 7.9% (305 residues) of cysteine residues on the d-Hml sequence had well-conserved positions in each motif. Coinciding with the development of phagocytic haemocytes, d-hml transcript was detected in late embryos and larvae. Its low-level expression in adult flies was induced by injury at any position on the body.

Amino Acid Sequence↗

PubMed2001

Olefin metathesis in supercritical carbon dioxide.

Liquid or supercritical carbon dioxide (scCO(2)) is a versatile reaction medium for ring-opening metathesis polymerization (ROMP) and ring-closing olefin metathesis (RCM) reactions using well-defined metal catalysts. The molybdenum alkylidene complex 1 and ruthenium carbenes 2 and 3 bearing PCy(3) or N-heterocyclic carbene ligands, respectively, can be used and are found to exhibit efficiency similar to that in chlorinated organic solvents. While compound 1 is readily soluble in scCO(2), complexes 2 and 3 behave like heterogeneous catalysts in this reaction medium. Importantly, however, the unique properties of scCO(2) provide significant advantages beyond simple solvent replacement. This pertains to highly convenient workup procedures both for polymeric and low molecular weight products, to catalyst immobilization, to reaction tuning by density control (RCM versus acyclic diene metathesis polymerization), and to applications of scCO(2) as a protective medium for basic amine functions. The latter phenomenon is explained by the reversible formation of the corresponding carbamic acid as evidenced by (1)H NMR data obtained in compressed CO(2). Together with its environmentally and toxicologically benign character, these unique physicochemical features sum up to a very attractive solvent profile of carbon dioxide for sustainable synthesis and production.

Journal Article↗

PubMed2001

A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum.

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

ATP-Binding Cassette Transporters↗

PubMed2001

YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids.

Like its mitochondrial homolog Oxa1p, the inner membrane protein YidC of Escherichia coli is involved in the integration of membrane proteins. We have analyzed individual insertion steps of the polytopic E. coli membrane protein MtlA targeted as ribosome-nascent chain complexes to inner membrane vesicles. YidC can accommodate at least the first two transmembrane segments of MtlA at the protein lipid interface and retain them even though the length of the nascent chain would amply allow insertion into membrane lipids. An even longer insertion intermediate of MtlA is described that still has the first transmembrane helix bound to YidC while the third contacts SecE and YidC during integration. Our findings suggest that YidC forms a contiguous integration unit with the SecYE translocon and functions as an assembly site for polytopic membrane proteins mediating the formation of helix bundles prior to their release into the membrane lipids.

Bacterial Proteins↗

PubMed2001

Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane.

Bacteria use several routes to target their exported proteins to the plasma membrane. The majority are exported through pores formed by SecY and SecE. Two different molecular machineries are used to target proteins to the SecYE translocon. Translocated proteins, synthesized as precursors with cleavable signal sequences, require cytoplasmic chaperones, such as SecB, to remain competent for posttranslational transport. In concert with SecB, SecA targets the precursors to SecY and energizes their translocation by its ATPase activity. The latter function involves a partial insertion of SecA itself into the SecYE translocon, a process that is strongly assisted by a couple of membrane proteins, SecG, SecD, SecF, YajC, and the proton gradient across the membrane. Integral membrane proteins, however, are specifically recognized by a direct interaction between their noncleaved signal anchor sequences and the bacterial signal recognition particle (SRP) consisting of Ffh and 4.5S RNA. Recognition occurs during synthesis at the ribosome and leads to a cotranslational targeting to SecYE that is mediated by FtsY and the hydrolysis of GTP. No other Sec protein is required for integration unless the membrane protein also contains long translocated domains that engage the SecA machinery. Discrimination between SecA/SecB- and SRP-dependent targeting involves the specificity of SRP for hydrophobic signal anchor sequences and the exclusion of SRP from nascent chains of translocated proteins by trigger factor, a ribosome-associated chaperone. The SecYE pore accepts only unfolded proteins. In contrast, a class of redox factor-containing proteins leaves the cell only as completely folded proteins. They are distinguished by a twin arginine motif of their signal sequences that by an unknown mechanism targets them to specific pores. A few membrane proteins insert spontaneously into the bacterial plasma membrane without the need for targeting factors and SecYE. Insertion depends only on hydrophobic interactions between their transmembrane segments and the lipid bilayer and on the transmembrane potential. Finally, outer membrane proteins of Gram-negative bacteria after having crossed the plasma membrane are released into the periplasm, where they undergo distinct folding events until they insert as trimers into the outer membrane. These folding processes require distinct molecular chaperones of the periplasm, such as Skp, SurA, and PpiD.

Adenosine Triphosphatases↗

PubMed2001

Effects of intravenous fat emulsions on lung function in patients with acute respiratory distress syndrome or sepsis.

OBJECTIVE: To investigate whether rapid or slowly infused intravenous fat emulsions affect the ratio of prostaglandin I2/thromboxane A2 in arterial blood, pulmonary hemodynamics, and gas exchange. DESIGN: Prospective, controlled, randomized, crossover study. SETTING: Operative intensive care unit of a university hospital. PATIENTS: Eighteen critically ill patients. Ten patients were stratified with severe sepsis, and eight patients had acute respiratory distress syndrome (ARDS). INTERVENTIONS: Patients were assigned randomly to receive intravenous fat emulsions (0.4 x resting energy expenditure) over 6 hrs (rapid fat infusion) or 24 hrs (slow fat infusion) along with a routine parenteral nutrition regimen, by using a crossover study design. MEASUREMENTS AND MAIN RESULTS: Systemic and pulmonary hemodynamics as well as gas exchange measurements were recorded via respective indwelling catheters. Arterial thromboxane B2 and 6-keto-prostaglandin-F1alpha plasma concentrations were obtained by radioimmunoassay, and 6-keto-prostaglandin-F1alpha/thromboxane B2 ratios (P/T ratios) were calculated. Data were collected immediately before and 6, 12, 18, and 24 hrs after onset of fat infusion. In the ARDS group, P/T ratio increased by rapid fat infusion. Concomitantly, pulmonary shunt fraction, alveolar-arterial oxygen tension difference [P(a-a)o2]/Pao2, and cardiac index increased as well, whereas pulmonary vascular resistance and Pao2/Fio2 declined. After slow fat infusion, a decreased P/T ratio was revealed. This was accompanied by decreased pulmonary shunt fraction, lowered P(a-a)o2/Pao2, and increased Pao2/Fio2. Correlations between plasma concentrations of 6-keto-prostaglandin-F1alpha or thromboxane B2 and measures of respiratory performance could be shown during rapid and slow fat infusion, respectively. In the sepsis group, the P/T ratio remained unchanged at either infusion rate, but pulmonary shunt fraction and P(a-a)o2/Pao2 decreased after rapid fat infusion, whereas Pao2/Fio2 increased. CONCLUSION: Pulmonary hemodynamics and gas exchange are related to changes of arterial prostanoid levels in ARDS patients, depending on the rate of fat infusion. In ARDS but not in sepsis patients clear of pulmonary organ failure, a changing balance of prostaglandin I2 and thromboxane A2 may modulate gas exchange, presumably via interference with hypoxic pulmonary vasoconstriction.

APACHE↗

PubMed2000

High and low affinity heparin-binding sites in the G domain of the mouse laminin alpha 4 chain.

G domains of the mouse laminin alpha 1 and alpha 4 chains consisting of its five subdomains LG1-LG5 were overexpressed in Chinese hamster ovary cells and purified by heparin chromatography. alpha 1LG1-LG5 and alpha 4LG1-LG5 eluted at NaCl concentrations of 0.30 and 0.47 m, respectively. In solid phase binding assays with immobilized heparin, half-maximal concentrations of 14 (alpha 1LG1-LG5) and 1.4 nm (alpha 4LG1-LG5) were observed. N-Glycan cleavage of alpha 4LG1-LG5 did not affect affinity to heparin. The affinity of alpha 4LG1-LG5 was significantly reduced upon denaturation with 8 m urea but could be recovered by removing urea. Chymotrypsin digestion of alpha 4LG1-LG5 yielded high and low heparin affinity fragments containing either the alpha 4LG4-LG5 or alpha 4LG2-LG3 modules, respectively. Trypsin digestion of heparin-bound alpha 4LG1-LG5 yielded a high affinity fragment of about 190 residues corresponding to the alpha 4LG4 module indicating that the high affinity binding site is contained within alpha 4LG4. Competition for heparin binding of synthetic peptides covering the alpha 4LG4 region with complete alpha 4LG1-LG5 suggests that the sequence AHGRL1521 is crucial for high affinity binding. Introduction of mutation of H1518A or R1520A in glutathione S-transferase fusion protein of the alpha 4LG4 module produced in Escherichia coli markedly reduced heparin binding activity of the wild type. When compared with the known structure of alpha 2LG5, this sequence corresponds to the turn connecting strands E and F of the 14-stranded beta-sheet sandwich, which is opposite to the proposed binding sites for calcium ion, alpha-dystroglycan, and heparan sulfate.

Animals↗

PubMed2000

Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine.

Mutations resulting in replacement of one obligate Gly residue within the repeating (Gly-Xaa-Yaa)(n) triplet pattern of the collagen type I triple helix are the major cause of osteogenesis imperfecta (OI). Phenotypes of OI involve fragile bones and range from mild to perinatal lethal. In this study, host-guest triple-helical peptides of the form acetyl-(Gly-Pro-Hyp)(3)-Zaa-Pro-Hyp-(Gly-Pro-Hyp)(4)-Gly-Gly-amide are used to isolate the influence of the residue replacing Gly on triple-helix stability, with Zaa = Gly, Ala, Arg, Asp, Glu, Cys, Ser, or Val. Any substitution for Zaa = Gly (melting temperature, T(m) = 45 degrees C) results in a dramatic destabilization of the triple helix. For Ala and Ser, T(m) decreases to approximately 10 degrees C, and for the Arg-, Val-, Glu-, and Asp-containing peptides, T(m) < 0 degrees C. A Gly --> Cys replacement results in T(m) < 0 degrees C under reducing conditions but shows a broad transition (T(m) approximately 19 degrees C) in an oxidizing environment. Addition of trimethylamine N-oxide increases T(m) by approximately 5 degrees C per 1 M trimethylamine N-oxide, resulting in stable triple-helix formation for all peptides and allowing comparison of relative stabilities. The order of disruption of different Gly replacements in these peptides can be represented as Ala </= Ser < CPO(red) < Arg < Val < Glu </= Asp. The rank of destabilization of substitutions for Gly in these Gly-Pro-Hyp-rich homotrimeric peptides shows a significant correlation with the severity of natural OI mutations in the alpha1 chain of type I collagen.

Amino Acid Substitution↗

PubMed2000

Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor.

Besides SecA and SecB, Escherichia coli cells possess a signal recognition particle (SRP) to target exported proteins to the SecY translocon. Using chemical and site-specific cross-linking in vitro, we show that SRP recognizes the first signal anchor sequence of a polytopic membrane protein (MtlA) resulting in cotranslational targeting of MtlA to SecY and phospholipids of the plasma membrane. In contrast, a possible interaction of SRP with the secretory protein pOmpA is prevented by the association of trigger factor with nascent pOmpA. Trigger factor also prevents SecA from binding to the first 125 amino acids of pOmpA when they are still associated with the ribosome. Under no experimental conditions was SecA found to interact with MtlA. Likewise, virtually no binding of trigger factor to ribosome-bound MtlA occurs even in the complete absence of SRP. Collectively, our results indicate that at the stage of nascent polypeptides, polytopic membrane proteins are selected by SRP for co-translational membrane targeting, whereas secretory proteins are directed into the SecA/SecB-mediated post-translational targeting pathway by means of their preferential recognition by trigger factor.

Adenosine Triphosphatases↗

PubMed2000

Combining a name algorithm with a capture-recapture method to retrieve cases of Turkish descent from a German population-based cancer registry.

An increasing proportion of the 2 million Turkish residents in Germany is reaching the age in which cancer becomes a common health problem. However, data on cancer incidence and survival among Turkish residents are lacking due to incomplete reporting of nationality in German cancer registries. In the population-based cancer registry of the Saarland, retrieval by reported nationality yielded only 38% (95% confidence interval (CI): 31-45%) of the estimated number of Turkish cases in the registry; furthermore, nationality information was found to be inaccurate, and completeness dependent on the vital status of cases. A newly developed algorithm based on family names retrieved 85% (95% CI: 79-90%) of Turkish cases. Combining the two sources in a capture-recapture approach yielded 91% (95% CI: 86-94%) of the estimated total number of Turkish cases. Hence, the name-based algorithm provides a new and attractive tool for valid registry-based cancer research among Turks in Germany.

Algorithms↗

PubMed2000

Growing importance of capitation in Switzerland.

Since its liberalization the Swiss health insurance market has shown risk selection activities of the insurance funds, which call for risk adjustment. Because risk selection continues to be profitable under the current risk adjustment formula, fast growing HMO and PPO plans are (mis)used to attract good risks rather than to contain costs. For fear of being replaced by one centralised fund, social health insurers are themselves proposing improvements of the risk adjustment formula, to be applied to funds. The revised formula proposed in this paper, applicable among funds for risk adjustment and to gate-keeping models to calculate fair capitation, explains 12.4% of the variance of health care expenditure, halves profits from risk selection, and uses only the (few) data that are available in Switzerland.

Adolescent↗

PubMed1999

Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.

Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.

Bacterial Outer Membrane Proteins↗

PubMed1999

Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal.

The ubiquitin/proteasome pathway is a highly conserved mechanism of proteolysis in all eukaryotes. Ubiquitin (Ub) is conjugated to proteolytic substrates through the sequential action of ubiquitin-activating (E1/Uba) and ubiquitin-conjugating (E2/Ubc) enzymes. The mechanism of substrate recognition and ubiquitination is an area of active investigation, and we have begun a site-directed mutagenesis approach to define the biochemical and biophysical properties of ubiquitin-conjugating enzymes. We have characterized a specific mutation in Ubc4 (Ubc4(P62S)) which was previously shown to cause a temperature-sensitive growth defect in several other Ubc's. Ubc4(P62S) was rapidly degraded in vivo, contributing to the loss of function. However, reconstitution experiments revealed that the catalytic activity of Ubc4(P62S) was reversibly inactivated at 37 degrees C, demonstrating that the primary defect of Ubc4(P62S) is its inability to form a ubiquitin thioester bond at high temperature. The in vivo defect is compounded by increased susceptibility of Ubc4(P62S) to degradation by the ubiquitin/proteasome pathway. We have exploited the temperature-dependent degradation of the P62S mutant to destabilize an otherwise stable test protein (glutathione S-transferase). The use of this mutant may provide a useful cis-acting temperature-inducible degradation signal.

Amino Acid Substitution↗

PubMed1999

Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.

Biological Transport↗