Hans Kleivdal

A Novel, Extraneuronal Role for Cyclin-dependent Protein Kinase 5 (CDK5) MODULATION OF cAMP-INDUCED APOPTOSIS IN RAT LEUKEMIA CELLS

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.

Outer membrane proteins of Methylococcus capsulatus (Bath)

Abstract Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of β-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins.

Native and recombinant FomA proteins were extracted by detergent from the cell envelopes of Fusobacterium nucleatum and Escherichia coli, and purified to near homogeneity by chromatography. Circular dichroism analysis revealed that the FomA protein consists predominantly of beta-sheets, in line with the previously proposed 16-stranded beta-barrel topology model. Results obtained by trypsin treatment of intact cells and cell envelopes of F. nucleatum, and from limited proteolysis of purified FomA protein, indicated that the N-terminal part of the FomA protein is not an integral part of the beta-barrel, but forms a periplasmic domain. Based on these results a new topology model is proposed for the FomA protein, where the C-terminal part forms a 14-stranded beta-barrel separate from the periplasmic N-terminal domain.

Topological investigations of the FomA porin from Fusobacterium nucleatum and identification of the constriction loop L6

Porin FomA in the outer membrane of Fusobacterium nucleatum is a trimeric protein, which exhibits permeability properties similar to that of the well-known enterobacterial diffusion porins. The proposed topology model of the FomA monomer depicts the beta-barrel motif typical of diffusion porins, consisting of 16 antiparallel beta-strands. To investigate the accuracy of the FomA model and assess the topological relationship with other porins, individual deletions of variable size in seven of the eight surface-exposed regions of the porin were genetically engineered. Deletions in the predicted loops L1 to L7 were tolerated by the FomA porins, as judged by a normal assembly in the outer membrane of Escherichia coli and a sustained pore-forming ability. Deletions in the largest proposed external region, loop L6, made the FomA porins considerably more permeable to antibiotics, indicating larger pore channels. The distinctly increased uptake rates and size exclusion limits displayed by the L6 deletion mutant porins, suggest that loop L6 folds back into the beta-barrel thereby constricting the native FomA channel. Thus, the position of the channel constriction loop appears to be shifted towards the C terminus in the FomA porin, as compared to the crystal structures of five non-specific diffusion porins.

The Fusobacterium nucleatum porin FomA possesses the general topology of the non-specific porins

FomA is a major non-specific porin of Fusobacterium nucleatum with no sequence similarity to other known porins. According to the topology model, the protein consists of 16 transmembrane beta-strands, connected by eight surface-exposed loops and seven periplasmic turns. In this study, the insertion mutagenesis approach was applied to probe the topology model. A Semliki Forest Virus (SFV) epitope was successfully inserted at 11 different sites of the FomA protein and a 6-aa insertion was successfully inserted at two different sites. Correct folding of the mutant proteins and proper incorporation into the outer membrane were assessed by heat modifiability and by an in vivo porin activity assay. Immunofluorescence microscopy analysis of intact cells, using mAbs directed against the inserted SFV epitope, revealed that three of the eight putative extracellular loops are indeed surface-exposed. Trypsin accessibility experiments confirmed the cell surface exposure of two additional loops. The results support the proposed topology model, showing that FomA possesses the general beta-barrel topology of the non-specific porins, with the interesting exception that the third loop does not seem to fulfil the role of a constriction loop.

A rapid solubility-optimized screening procedure for recombinant subtilisins in E. coli

Subtilisins and other serine proteases are extensively used in the detergent, leather and food industry, and frequently under non-physiological conditions. New proteases with improved performance at extreme temperatures and in the presence of chemical additives may have great economical potential. The increasing availability of genetic sequences from different environments makes homology-based screening an attractive strategy for discovery of new proteases. A prerequisite for large-scale screening of protease-encoding sequences is an efficient screening procedure. We have developed and implemented a screening procedure that encompasses cloning of candidate sequences into multiple expression vectors, cytoplasmic expression in E. coli, and a casein-based functional screen. The procedure is plate-format compatible and can be completed in only four days, starting from the gene of interest in a suitable cloning vector. The expression vector suite includes six vectors with combinations of maltose-binding protein (MBP) or the small ubiquitin-related modifier (SUMO) for increased solubility, and polyhistidine tags for downstream purification. We used enhanced green fluorescent protein and four Bacilli subtilisins to validate the screening procedure and our results show that proteins were expressed, soluble and active. Interestingly, the highest activities were consistently achieved with either MBP or SUMO fusions, thus demonstrating the merit of including solubility tags. In conclusion, the results demonstrate that our approach can be used to efficiently screen for new subtilisins, and suggest that the approach may also be used to screen for proteins with other activities.