SFRP1

A team of researchers reconstructed long-extinct proteins of a UV protection system of cyanobacteria. The surprising result: the proteins were already compatible with each other when they first met. This discovery expands the knowledge horizon on the rules of evolution. Proteins are the key players for virtually all molecular processes within the cell. To fulfil their diverse functions, they have to interact with other proteins. Such protein-protein interactions are mediated by highly complementary surfaces, which typically involve many amino acids that are positioned precisely to produce a tight, specific fit between two proteins. However, comparatively little is known about how such interactions are created during evolution. Classical evolutionary theory suggests that any new biological feature involving many components (like the amino acids that enable an interaction between proteins) evolves in a stepwise manner. According to this concept, each tiny functional improvement is driven by the power of natural selection because there is some benefit associated with the feature. However, whether protein-protein interactions also always follow this trajectory was not entirely known. Using a highly interdisciplinary approach, an international team led by Max Planck researcher Georg Hochberg from the Terrestrial Microbiology in Marburg have now shed new light on this question. Their study provides definitive evidence that highly complementary and biologically relevant protein-protein interactions can evolve entirely by chance. Proteins cooperate in a photoprotection system The research team made their discovery in a biochemical system that microbes use to adapt to stressful light conditions. Cyanobacteria use sunlight to produce their own food through photosynthesis. Since much light damages the cell, cyanobacteria have evolved a mechanism known as photoprotection: if light intensities become dangerously high, a light intensity sensor named Orange Carotenoid Protein (OCP) changes its shape. In this activated form, OCP protects the cell by converting excess light energy into harmless heat. In order to return into its original state, some OCPs depend on a second protein: The Fluorescence Recovery Protein (FRP) binds to activated OCP1 and strongly accelerates its recovery. 'Our question was: Is it possible that the surfaces that allow these two proteins to form a complex evolved entirely by accident, rather than through direct natural selection?' says Georg Hochberg. 'The difficulty is that the end result of both processes looks the same, so we usually cannot tell why the amino acids required for some interaction evolved -- through natural selection for the interaction or by chance. To tell them apart, we would need a time machine to witness the exact moment in history these mutations occurred, 'Georg Hochberg explains. Luckily, recent breakthroughs in molecular and computational biology has equipped Georg Hochberg and his team with a laboratory kind of time machine: ancestral sequence reconstruction. In addition, the light protection system of cyanobacteria, which is under study in the group of Thomas Friedrich from Technische Universität Berlin since many years, is ideal for studying the evolutionary encounter of two protein components. Early cyanobacteria acquired the FRP proteins from a proteobacterium by horizontal gene transfer. The latter had no photosynthetic capacity itself and did not possess the OCP protein. To work out how the interaction between OCP1 and FRP evolved, graduate student Niklas Steube inferred the sequences of ancient OCPs and FRPs that existed billions of years ago in the past, and then resurrected these in the laboratory. After translation of the amino acid sequences into DNA he produced them using E. coli bacterial cells in order to be able to study their molecular properties. A fortunate coincidence The Berlin team then tested whether ancient molecules could form an interaction. This way the scientists could retrace how both protein partners got to know each other. 'Surprisingly, the FRP from the proteobacteria already matched the ancestral OCP of the cyanobacteria, before gene transfer had even taken place. The mutual compatibility of FRP and OCP has thus evolved completely independently of each other in different species, says Thomas Friedrich. This allowed the team to prove that their ability to interact must have been a happy accident: selection could not plausibly have shaped the two proteins' surfaces to enable an interaction if they had never met each other. This finally proved that such interactions can evolve entirely without direct selective pressure. 'This may seem like an extraordinary coincidence,' Niklas Steube says. 'Imagine an alien spaceship landed on earth and we found that it contained plug-shaped objects that perfectly fit into human-made sockets. But despite the perceived improbability, such coincidences could be relatively common. But in fact, proteins often encounter a large number of new potential interaction partners when localisation or expression patterns change within the cell, or when new proteins enter the cell through horizontal gene transfer.' Georg Hochberg adds, 'Even if only a small fraction of such encounters ends up being productive, fortuitous compatibility may be the basis of a significant fraction of all interactions we see inside cells today. Thus, as in human partnerships, a good evolutionary match could be the result of a chance meeting of two already compatible partners.'

Morphogens are the secreted molecules that pattern embryonic tissues. These molecules are important not only for the embryo during development, but also for the adult during tissue repair. However, the way these morphogens are distributed to ensure patterning occurs is still not fully understood. Using a combination of experiments and mathematical modeling, researchers learned more about the role morphogens play in tissue patterning. Their results are relevant to medical applications, such as drug design. Morphogens are molecules that travel from biological cell to cell to pattern tissues in the embryo. These molecules are important not only for the embryo during development, but also for the adult during tissue repair. However, the way these morphogens are distributed to ensure patterning occurs is still not fully understood. Using a combination of experiments and mathematical modeling, a research team from the University of Tokyo and their international collaborators has learned more about the role morphogens play in tissue patterning. The results are relevant to medical applications, such as drug design. The team's findings are published in the journal eLife. The Wnt morphogen has emerged as a key regulator of heart development in vertebrates. These Wnt proteins are molecules that play an important role in cell development. However, it is still generally unclear to scientists exactly how Wnt regulates heart development. There are differences among the vertebrate classes, as well as redundancy in some species. However, scientists can study how Wnt regulates heart development in the Xenopus, an aquatic frog that is native to sub-Saharan Africa. The Xenopus, with its lungs and three-chamber heart, is cost-effective and useful to scientists in their study of human diseases. In Xenopus heart development, scientists have already established that the Wnt6 morphogen is sent out by the epidermis, those outer layers of cells that make up the skin, to pattern the cardiogenic mesoderm, which is the group of cells in the embryo that will form the heart. From this patterning, a relatively thin pericardium (the membrane around the heart) and a broad myocardium (the heart's muscular tissue) develop. Scientists are still working to better understand how the Wnt6 morphogen distribution is regulated to ensure reproducible positioning of the pericardium and myocardium in the cardiogenic mesoderm. "It is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when that patterning concerns differentiation of thin tissues." said Takayoshi Yamamoto, an assistant professor at the University of Tokyo and the first author and corresponding on the paper. Scientists do know that in early embryo development, the range of Wnt8 morphogen signaling is precisely regulated heparan sulfate and secreted Wnt binding proteins, including Frzb (which is also known as sFRP3). Heparan sulfate is a carbohydrate that is important is embryo development. Wnt signaling is one of the main processes by which tissue takes shape during the development of the embryo. The research team wondered whether mechanisms similar to those that operate in early embryos also regulate distribution of Wnt6 morphogen in the cardiogenic mesoderm. The Wnt receptor, Frizzled7, is essential for heart development. The expression of Frizzled7 is increased by Wnt signaling in the development of the nervous system in the Xenopus and in the development of human embryonic carcinoma cells, but there are no such reports in heart development. So the research team focused their study to analyze the way Wnt signaling occurs in the development of the heart, focusing on the extracellular components -- the Frizzled7 cell-surface receptor, sFRP1 (an inhibitor of Wnt6 that can also travel from cell to cell) and heparan sulfate. "With a combination of experiments and mathematical modeling, this receptor-feedback appears essential to shape a steep gradient of Wnt signaling. In addition, computer simulation revealed that this feedback imparts robustness against variations of Wnt ligand production and allows the system to reach a steady state quickly," said Yamamoto. Wnt6 and sFRP1 molecules not only regulate normal heart development in the embryo, but also regulate repair and regeneration after heart muscle injury, such as in the case of a myocardial infarction, or heart attack. "Our findings will be relevant to medical applications, for example, for drug design, since cell-surface molecules such as Frizzled or a specific modification of heparan sulfate or even the secreted molecule sFRP1, generally provide better drug targets than molecules inside cells. To reveal the precise regulation of morphogens and to consider medical applications, regulatory mechanisms of these components must be investigated further," said Yamamoto. The research was conducted in collaboration with researchers at the University of Aberdeen in the U.K.