TWISTED DWARF1 determines downstream locations of auxin efflux transporters by adjusting actin cytoskeleton dynamics and mediates NPA action on these in an acti

Inhibition of autophagic proteolysis by hypoosmotic or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; however, the upstream osmosensing and signaling events are unknown. These were studied in the intact perfused rat liver with a preserved in situ environment of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an activation of Src (but not FAK), Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP. Src inhibition by PP-2 prevented hypoosmotic MAP kinase activation, indicating that the integrin/Src system is located upstream in the osmosignaling toward p38MAPK and Erks. Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 also prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These inhibitors, however, did not affect swelling-independent proteolysis inhibition by phenylalanine. In line with a role of p38MAPK in triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide blunted RVD in response to hypoosmotic cell swelling. The data identify integrins and Src as upstream events in the osmosignaling toward MAP kinases, proteolysis, and RVD. They further point to a role of integrins as osmo- and mechanosensors in the intact liver, which may provide a link between cell volume and cell function.

On a short term basis, canalicular secretion is under control of the hepatocellular hydration state, substrates, cytokines, toxins, and hormones. Regulation occurs at the level of substrate availability, covalent modification of transporters, and their regulated exocytic insertion into or endocytic retrieval from the membrane. A variety of signal transduction pathways involving the activation of mitogen-activated protein kinases, protein kinases A and C, participates in these processes. However, much has still to be learned about the crosstalk of different signaling systems and their molecular targets that determine the outcome for canalicular secretion.

The extracellular domain of the human interleukin‐6 (IL‐6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus‐infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL‐6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 × 10⁴l · mol⁻¹· cm⁻¹ was calculated from the ultraviolet spectrum of the soluble IL‐6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N‐glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41–44 kDa. As expected, the soluble human IL‐6 receptor bound human ¹²⁵I‐labeled IL‐6 with low affinity (K_d= 500 pM). Furthermore, the binding of soluble human IL‐6 receptor to immobilized IL‐6 was studied using real‐time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL‐6 (HepG2‐IL‐6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL‐6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1–67 and 68–143 of the soluble IL‐6 receptor, respectively. The plasma clearance of soluble ¹²⁵I‐labeled IL‐6 receptor in the absence and presence of IL‐6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL‐6 receptor/IL‐6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble ¹²⁵I‐labeled IL‐6 receptor, as well as complexes with IL‐6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys, Subsequently, the radioactivity appeared in the gut content.

Understanding enzyme stability and activity in extremophilic organisms is of great biotechnological interest, but many questions are still unsolved. Using 2-deoxy-D-ribose-5-phosphate aldolase (DERA) as model enzyme, we have evaluated structural and functional characteristics of different orthologs from psychrophilic, mesophilic and hyperthermophilic organisms. We present the first crystal structures of psychrophilic DERAs, revealing a dimeric organization resembling their mesophilic but not their thermophilic counterparts. Conversion into monomeric proteins showed that the native dimer interface contributes to stability only in the hyperthermophilic enzymes. Nevertheless, introduction of a disulfide bridge in the interface of a psychrophilic DERA did confer increased thermostability, suggesting a strategy for rational design of more durable enzyme variants. Constraint network analysis revealed particularly sparse interactions between the substrate pocket and its surrounding α-helices in psychrophilic DERAs, which indicates that a more flexible active center underlies their high turnover numbers.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is used in organic synthesis for the enantioselective reaction between acetaldehyde and a broad range of other aldehydes as acceptor molecules. Nevertheless, its application is hampered by a poor tolerance towards high concentrations of acetaldehyde, its natural substrate. While numerous studies have been performed searching for new, more acetaldehyde-resistant DERAs, the mechanism underlying this deactivation process has remained elusive. By using NMR spectroscopy on both the protein and the small-molecule scale, we could show that a reaction product binds to the inner part of the enzyme, and that this effect can be partly reversed via heating. The crystal structure of DERA before and after acetaldehyde incubation was determined at high resolution, revealing a covalently bound reaction product bridging the catalytically active lysine (K167) to a nearby cysteine (C47) in the deactivated enzyme. A reaction mechanism is proposed where crotonaldehyde as the aldol product of two acetaldehyde molecules after water elimination forms a Schiff base with the lysine side chain, followed by Michael addition of the cysteine thiol group to the C_β atom of the inhibitor. In support of this mechanism, direct incubation of DERA with crotonaldehyde results in a more than 100-fold stronger inhibition, compared to acetaldehyde, whereas mutation of C47 gives rise to a fully acetaldehyde-resistant DERA. Thus this variant appears perfectly suited for synthetic applications. A similar diagnostic and preventive strategy should be applicable to other biocatalysts suffering from mechanism-based inhibition by a reactive substrate, a condition that may be more common than currently appreciated in biotechnology.