Lipps, Georg
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The monomeric archaeal primase from Nanoarchaeum equitans harbours the features of heterodimeric archaeoeukaryotic primases and primes sequence-specifically
The marine thermophilic archaeon Nanoarchaeum equitans possesses a monomeric primase encompassing the conserved domains of the small catalytic and the large regulatory subunits of archaeoeukaryotic heterodimeric primases in one protein chain. The recombinant protein primes on templates containing a triplet with a central thymidine, thus displaying a pronounced sequence specificity typically observed with bacterial type primases only. The N. equitans primase (NEQ395) is a highly active primase enzyme synthesizing short RNA primers. Termination occurs preferentially at about nine nucleotides, as determined by HPLC analysis and confirmed with mass spectrometry. Possibly, the compact monomeric primase NEQ395 represents the minimal archaeoeukaryotic primase and could serve as a functional and structural model of the heterodimeric archaeoeukaryotic primases, whose study is hindered by engagement in protein assemblies and rather low activity.
Stringent primer termination by an archaeo-eukaryotic DNA primase
Priming of single stranded templates is essential for DNA replication. In recent years, significant progress was made in understanding how DNA primase fulfils this fundamental function, particularly with regard to the initiation. Equally intriguing is the unique property of archeao-eukaryotic primases to terminate primer formation at a well-defined unit length. The apparent ability to “count” the number of bases incorporated prior to primer release is not well understood, different mechanisms having been proposed for different species. We report a mechanistic investigation of primer termination by the pRN1 primase from Sulfolobus islandicus. Using an HPLC-based assay we determined structural features of the primer 5′-end that are required for consistent termination. Mutations within the unstructured linker connecting the catalytic domain to the template binding domain allowed us to assess the effect of altered linker length and flexibility on primer termination.
Characterization of the housekeeping sortase from the human pathogen Propionibacterium acnes - first investigation of a class F sortase
Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications. © 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
Biological properties of extracellular vesicles and their physiological functions
Stable and selective permeable hydrogel microcapsules for high-throughput cell cultivation and enzymatic analysis
A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template
Recent advances in understanding bacterial and archaeoeukaryotic primases
DNA replication in all forms of life relies upon the initiation of synthesis on a single strand template by formation of a short oligonucleotide primer, which is subsequently elongated by DNA polymerases. Two structurally distinct classes of enzymes have evolved to perform this function, namely the bacterial DnaG-type primases and the Archaeal and Eukaryotic primases (AEP). Structural and mechanistic insights have provided a clear understanding of the role of the different domains of these enzymes in the context of the replisome and recent work sheds light upon primase-substrate interactions. We herein review the emerging picture of the primase mechanism on the basis of the structural knowledge obtained to date and propose future directions of this essential aspect of DNA replication.
Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine
Recent research has demonstrated that all body fluids assessed contain substantial amts. of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes contg. different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivs. of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiol. and pathophysiol. conditions. Contg. cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their phys. functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clin., academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technol. (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.