Regulation, evolution and consequences of cotranslational protein complex assembly.

Most proteins assemble into complexes, which are involved in almost all cellular processes. Thus it is crucial for cell viability that mechanisms for correct assembly exist. The timing of assembly plays a key role in determining the fate of the protein: if the protein is allowed to diffuse into the crowded cellular milieu, it runs the risk of forming non-specific interactions, potentially leading to aggregation or other deleterious outcomes. It is therefore expected that strong regulatory mechanisms should exist to ensure efficient assembly. In this review we discuss the cotranslational assembly of protein complexes and discuss how it occurs, ways in which it is regulated, potential disadvantages of cotranslational interactions between proteins and the implications for the inheritance of dominant-negative genetic disorders

Mapping Rora expression in resting and activated CD4+ T cells.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it might also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched towards the C-termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur prior to assembly. Using high-throughput imaging of protein homomers in vivo in E. coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization

Assembly in the translation milieu imposes evolutionary constraints on homomeric proteins

<p><b>Supplementary Video 1. Simulation</b> <b>of Tet@N with short linker. </b>For all movies the red segment is the Tet, and yellow is the YFP beta-barrel. Both cotranslational folding and misassembly take place once the Tet appear outside of the ribosome tunnels.</p><p><br></p><p> </p><p><b>Supplementary Video 2. Simulation</b> <b>of Tet@C with short linker. </b>There was no cotranslational assembly as the Tet of the leading ribosome leaves the ribosomal tunnel prior to the translation of the second ribosome, which allows it time to diffuse before the second Tet leaves the ribosome tunnel. </p> <p><b> </b></p> <p><b>Supplementary Video 3. Simulation</b> <b>of Tet@N with long linker. </b>Cotranslational assembly takes place, but not misassembly. Similarly to movies S1-S2, the red segment is the Tet and the yellow is the YFP reporter-gene, the cyan segment is the long-linker. The less frequent misassembly events fit the observed <i>in vivo and in vitro</i> data. </p> <p><b> </b></p

The symbiotic magnetic-sensing hypothesis: do Magnetotactic Bacteria underlie the magnetic sensing capability of animals?

Abstract The ability to sense Earth’s magnetic field has evolved in various taxa. However, despite great efforts to find the ‘magnetic-sensor’ in vertebrates, the results of these scientific efforts remain inconclusive. A few decades ago, it was found that bacteria, known as magnetotactic bacteria (MTB), can move along a magnetic field using nanometric chain-like structures. Still, it is not fully clear why these bacteria evolved to have this capacity. Thus, while for MTB the ‘magnetic-sensor’ is known but the adaptive value is still under debate, for metazoa it is the other way around. In the absence of convincing evidence for any ‘magnetic-sensor’ in metazoan species sensitive to Earth’s magnetic field, we hypothesize that a mutualism between these species and MTB provides one. In this relationship the host benefits from a magnetotactic capacity, while the bacteria benefit a hosting environment and dispersal. We provide support for this hypothesis using existing literature, demonstrating that by placing the MTB as the ‘magnetic-sensor’, previously contradictory results are now in agreement. We also propose plausible mechanisms and ways to test the hypothesis. If proven correct, this hypothesis would shed light on the forces driving both animal and bacteria magnetotactic abilities

Structure and Kinetic Stability of the p63 Tetramerization Domain

The p53 family of transcription factors—comprising p53, p63 and p73—plays an important role in tumor prevention and development. Essential to their function is the formation of tetramers, allowing cooperative binding to their DNA response elements. We solved crystal structures of the human p63 tetramerization domain, showing that p63 forms a dimer of dimers with D2 symmetry composed of highly intertwined monomers. The primary dimers are formed via an intramolecular β-sheet and hydrophobic helix packing (H1), a hallmark of all p53 family members. Like p73, but unlike p53, p63 requires a second helix (H2) to stabilize the architecture of the tetramer. In order to investigate the impact of structural differences on tetramer stability, we measured the subunit exchange reaction of p53 family homotetramers by nanoflow electrospray mass spectrometry. There were differences in both the kinetics and the pattern of the exchange reaction, with the p53 and p63 tetramers exhibiting much faster exchange kinetics than p73. The structural similarity between p63 and p73 rationalizes previous observations that p63 and p73 form mixed tetramers, and the kinetic data reveal the dissociation of the p73 homotetramers as the rate-limiting step for heterotetramer formation. Differential stability of the tetramers may play an important role in the cross talk between different isoforms and regulation of p53, p63 and p73 function in the cell cycle

Interaction of the p53 DNA-Binding Domain with Its N-Terminal Extension Modulates the Stability of the p53 Tetramer

The tetrameric tumor suppressor p53 plays a pivotal role in the control of the cell cycle and provides a paradigm for an emerging class of oligomeric, multidomain proteins with structured and intrinsically disordered regions. Many of its biophysical and functional properties have been extrapolated from truncated variants, yet the exact structural and functional role of certain segments of the protein is unclear. We found from NMR and X-ray crystallography that the DNA-binding domain (DBD) of human p53, usually defined as residues 94–292, extends beyond these domain boundaries. Trp91, in the hinge region between the disordered proline-rich N-terminal domain and the DBD, folds back onto the latter and has a cation–π interaction with Arg174. These additional interactions increase the melting temperature of the DBD by up to 2 °C and inhibit aggregation of the p53 tetramer. They also modulate the dissociation of the p53 tetramer. The absence of the Trp91/Arg174 packing presumably allows nonnative DBD–DBD interactions that both nucleate aggregation and stabilize the interface. These data have important implications for studies of multidomain proteins in general, highlighting the fact that weak ordered–disordered domain interactions can modulate the properties of proteins of complex structure