Rafal Wieczorek

A novel potassium channel in skeletal muscle mitochondria

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

Ser-His catalyses the formation of peptides and PNAs

The dipeptide seryl‐histidine (Ser‐His) catalyses the condensation of esters of amino acids, peptide fragments, and peptide nucleic acid (PNA) building blocks, bringing to the formation of peptide bonds. Di‐, tri‐ or tetra‐peptides can be formed with yields that vary from 0.5% to 60% depending on the nature of the substrate and on the conditions. Other simpler peptides as Gly‐Gly, or Gly‐Gly‐Gly are also effective, although less efficiently. We discuss the results from the viewpoint of primitive chemistry and the origin of long macromolecules by stepwise fragment condensations.

Formation of RNA Phosphodiester Bond by Histidine-Containing Dipeptides

A new scenario for prebiotic formation of nucleic acid oligomers is presented. Peptide catalysis is applied to achieve condensation of activated RNA monomers into short RNA chains. As catalysts, L‐dipeptides containing a histidine residue, primarily Ser‐His, were used. Reactions were carried out in selforganised environment, a water‐ice eutectic phase, with low concentrations of reactants. Incubation periods up to 30 days resulted in the formation of short oligomers of RNA. During the oligomerisation, an active intermediate (dipeptide–mononucleotide) is produced, which is the reactive species. Details of the mechanism and kinetics, which were elucidated with a set of control experiments, further establish that the imidazole side chain of a histidine at the carboxyl end of the dipeptide plays a crucial role in the catalysis. These results suggest that this oligomerisation catalysis occurs by a transamination mechanism. Because peptides are much more likely products of spontaneous condensation than nucleotide chains, their potential as catalysts for the formation of RNA is interesting from the origin‐of‐life perspective. Finally, the formation of the dipeptide–mononucleotide intermediate and its significance for catalysis might also be viewed as the tell‐tale signs of a new example of organocatalysis.

Open questions in origin of life: experimental studies on the origin of nucleic acids and proteins with specific and functional sequences by a chemical synthetic biology approach.

In this mini-review we present some experimental approaches to the important issue in the origin of life, namely the origin of nucleic acids and proteins with specific and functional sequences. The formation of macromolecules on prebiotic Earth faces practical and conceptual difficulties. From the chemical viewpoint, macromolecules are formed by chemical pathways leading to the condensation of building blocks (amino acids, or nucleotides) in long-chain copolymers (proteins and nucleic acids, respectively). The second difficulty deals with a conceptual problem, namely with the emergence of specific sequences among a vast array of possible ones, the huge “sequence space”, leading to the question “why these macromolecules, and not the others?” We have recently addressed these questions by using a chemical synthetic biology approach. In particular, we have tested the catalytic activity of small peptides, like Ser-His, with respect to peptide- and nucleotides-condensation, as a realistic model of primitive organocatalysis. We have also set up a strategy for exploring the sequence space of random proteins and RNAs (the so-called “never born biopolymer” project) with respect to the production of folded structures. Being still far from solved, the main aspects of these “open questions” are discussed here, by commenting on recent results obtained in our groups and by providing a unifying view on the problem and possible solutions. In particular, we propose a general scenario for macromolecule formation via fragment-condensation, as a scheme for the emergence of specific sequences based on molecular growth and selection.

On prebiotic ecology, supramolecular selection and autopoiesis.

Keywords Originoflife.Autopoiesis.Prebioticecology.RNAworldcritique.LipidworldThe existence of many “worlds” in origin-of-life literature; such as the “lipid world” (Segre etal.2001),the“iron-sulfur world ”(Wachtershauser1992),the“aromaticworld”(Ehrenfreundetal. 2006) and others, leads to the impression that there are many competing hypothesis for theorigin of life. However, if one postulates that a concrete theory for the origin of life shouldprovide a plausible pathway from the stage of prebiotic soup to the stage of life in a series oflogical steps, then none of the above mentioned “worlds” comply with this requirement. Mostsimply provide us with new or alternative ways of making components of the prebiotic soup,whether monomeric or polymeric.There is only one origin-of-life theory which complies with the above stated requirement:the so called “RNA world” theory (Gilbert 1986). A brief, modern recapitulation of theRNA-world theory goes something like this: On an early Earth there was a prebiotic soupstage in which, in some aqueous environment, there was an abundance of various organicmolecules, including nucleotide components. The complex interactions of these molecules,at some point, allowed for the emergence of RNA chains and later on a self-replicating RNAmolecule. Once such a molecule was present it made many copies of itself. Due to non-perfect replication, some of the molecules were different and thus behaved differently in theenvironment (for example they were better replicators), thus paving the way for hereditaryfeatures, environmental selection and therefore evolution. Some of these RNAs (namelyribozymes) possessed catalytic activities. RNA molecules which were encapsulated insidelipid vesicles were advantaged over those that were not, principally thanks to protectionfrom the outside environment. Later on those that were able to catalyse synthesis of theirown lipid vesicles prevailed over those that could not. At this stage the macromolecularconglomerate started resembling modern cells and the RNA in these “cells” performed bothgenetic and enzymatic functions. At some point, the RNA protocell “invented” DNA for

Bottom–Up Protocell Design: Gaining Insights in the Emergence of Complex Functions

All contemporary living cells are a collection of self-assembled molecular elements that by themselves are non-living but through the creation of a network exhibit the emergent properties of self-maintenance, self-reproduction, and evolution. Protocells are chemical systems that should mimic cell behavior and their emergent properties through the interactions of their components. For a functional protocell designed bottom-up, three fundamental elements are required: a compartment, a reaction network, and an information system. Even if the functions of protocell components are very simplified compared to those of modern cells, realizing a system with true inter-connection and inter-dependence of all the functions should lead to emergent properties. However, none of the currently studied systems have yet reached the threshold level necessary to be considered alive. This chapter will discuss the on-going research that aims at creating artificial cells assembled from a collection of smaller components, i.e., protocell systems from bottom-up designs.