Structural behaviour and gene delivery in complexes formed between DNA and arginine-containing peptide amphiphiles

We describe in depth the structure of complexes formed between DNA and two classes of arginine-containing peptide amphiphiles, namely, the lipopeptide PRW–C16 (P = proline, R = arginine, W = tryptophan, C16 = C16 : 0 alkyl chain) and the bolaamphiphile RFL4FR (R = arginine, F = phenylalanine, L = leucine). A combination of X-ray and neutron scattering provided unprecedented insights into the local structure of these complexes. Lipopeptide-based complexes self-assembled into layered structures with large-scale fractal features, hosting DNA in the interstices. Bola-amphiphile scaffolds were characterized by planar structures with DNA strands presumably sandwiched in-between peptide nanotapes. Importantly, complexation did not affect the structural integrity of DNA in either of the two complexes. The bolaamphiphile conjugates displayed high levels of molecular ordering in contrast to the liquid-crystalline features observed in lipopeptide assemblies. Peptide–DNA complexes were assessed for their potential as a means to deliver the reporter vector pEGFP-N1 into SW480 human colon carcinoma cells. Successfully transfected cells expressed green fluorescent protein. The potentiating effect of PRW–C16 on the cellular uptake of ectopic DNA was found to be much greater than that observed with RFL4FR. In contrast to the bolaamphiphile-based conjugate, the liquid-crystalline nature of the lipopeptide complex is likely to play a key role in DNA release and transfection efficiency since these weakly bound structures require lower energy expenditure during disassembly and load release

The 20s proteasome of streptomyces coelicolor

20S proteasomes were purified from Streptomyces coelicolor A3(2) and shown to be built from one alpha-type subunit (PrcA) and one beta-type subunit (PrcB). The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the Streptomyces metabolite lactacystin. Characterization of the structural genes revealed an operon-like gene organization (prcBA) similar to Rhodococcus and Mycobacterium spp. and showed that the beta subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly. The upstream DNA region contains the conserved orf7 and an AAA ATPase gene (arc). [References: 48

Structural analysis of the 6 kb cryptic plasmid pFAJ26OO from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli Rhodococcus shuttle vectors

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cm-r derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions

Subunit topology of the rhodococcus proteasome

The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two alpha-type and two beta-type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the alpha- and beta-subunits in all. possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details. [References: 30

Eubacterial proteasomes

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependentpathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure. [References: 50

Characterization of arc, a divergent member of the aaa atpase family from rhodococcus erythropolis

A gene encoding a AAA ATPase was discovered in the 5' region of the second operon of 20 S proteasome subunits in the nocardioform actinomycete Rhodococcus erythropolis NI86/21. The gene was cloned and expressed in Escherichia coli. The protein, ARC (AAA ATPase forming Ring-shaped Complexes), is a divergent member of the AAA family. The deduced product of the are gene is 591 residues long (66 kDa). The purified protein possesses a low, N-ethylmaleimide-sensitive ATPase activity and forms rings of six subunits, arranged symmetrically around a central opening or cavity. Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in ''end-on'' orientation at 1.9 nm resolution. (C) 1998 Academic Press Limited. [References: 57

The first characterization of a eubacterial proteasome - the 2os complex of rhodococcus

Background: The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). Results: We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05 % SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. Conclusions: The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life. [References: 41