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A considerably small fraction of approximately 60–100 proteins of all chloroplast proteins are encoded by the plastid genome. Many of these proteins are major subunits of complexes with central functions within plastids. In comparison with other subcellular compartments and bacteria, many steps of chloroplast protein biogenesis are not well understood. We report here on the first study of chloroplast-localised trigger factor. In bacteria, this molecular chaperone is known to associate with translating ribosomes to facilitate the folding of newly synthesized proteins. Chloroplast trigger factors of the unicellular green algae Chlamydomonas reinhardtii and the vascular land plant Arabidopsis thaliana were characterized by biophysical and structural methods and compared to the Escherichia coli isoform. We show that chloroplast trigger factor is mainly monomeric and displays only moderate stability against thermal unfolding even under mild heat-stress conditions. The global shape and conformation of these proteins were determined in solution by small-angle X-ray scattering and subsequent ab initio modelling. As observed for bacteria, plastidic trigger factors have a dragon-like structure, albeit with slightly altered domain arrangement and flexibility. This structural conservation despite low amino acid sequence homology illustrates a remarkable evolutionary robustness of chaperone conformations across various kingdoms of life.

Biochemical processes in chloroplasts are important for virtually all life forms. Tight regulation of protein homeostasis and the coordinated assembly of protein complexes, composed of both imported and locally synthesized subunits, are vital to plastid functionality. Protein biogenesis requires the action of cotranslationally acting molecular chaperones. One such chaperone is trigger factor (TF), which is known to cotranslationally bind most newly synthesized proteins in bacteria, thereby assisting their correct folding and maturation. However, how these processes are regulated in chloroplasts remains poorly understood. We report here functional investigation of chloroplast-localized TF (TIG1) in the green alga (Chlamydomonas reinhardtii) and the vascular land plant Arabidopsis (Arabidopsis thaliana). We show that chloroplastic TIG1 evolved as a specialized chaperone. Unlike other plastidic chaperones that are functionally interchangeable with their prokaryotic counterpart, TIG1 was not able to complement the broadly acting ortholog in Escherichia coli. Whereas general chaperone properties such as the prevention of aggregates or substrate recognition seems to be conserved between bacterial and plastidic TFs, plant TIG1s differed by associating with only a relatively small population of translating ribosomes. Furthermore, a reduction of plastidic TIG1 levels leads to deregulated protein biogenesis at the expense of increased translation, thereby disrupting the chloroplast energy household. This suggests a central role of TIG1 in protein biogenesis in the chloroplast.

Vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress. Competing Interest Statement The authors have declared no competing interest.

Glioblastoma (GBM) is the most difficult brain cancer to treat, and photodynamic therapy (PDT) is emerging as a complementary approach to improve tumor eradication. Neuropilin-1 (NRP-1) protein expression plays a critical role in GBM progression and immune response. Moreover, various clinical databases highlight a relationship between NRP-1 and M2 macrophage infiltration. In order to induce a photodynamic effect, multifunctional AGuIX®-design nanoparticles were used in combination with a magnetic resonance imaging (MRI) contrast agent, as well as a porphyrin as the photosensitizer molecule and KDKPPR peptide ligand for targeting the NRP-1 receptor. The main objective of this study was to characterize the impact of macrophage NRP-1 protein expression on the uptake of functionalized AGuIX®-design nanoparticles in vitro and to describe the influence of GBM cell secretome post-PDT on the polarization of macrophages into M1 or M2 phenotypes. By using THP-1 human monocytes, successful polarization into the macrophage phenotypes was argued via specific morphological traits, discriminant nucleocytoplasmic ratio values, and different adhesion abilities based on real-time cell impedance measurements. In addition, macrophage polarization was confirmed via the transcript-level expression of TNFα, CXCL10, CD-80, CD-163, CD-206, and CCL22 markers. In relation to NRP-1 protein over-expression, we demonstrated a three-fold increase in functionalized nanoparticle uptake for the M2 macrophages compared to the M1 phenotype. The secretome of the post-PDT GBM cells led to nearly a three-fold increase in the over-expression of TNFα transcripts, confirming the polarization to the M1 phenotype. The in vivo relationship between post-PDT efficiency and the inflammatory effects points to the extensive involvement of macrophages in the tumor zone.

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring. Influenza infection during pregnancy can affect health of offspring but it is not clear how this affects immune responses. Here the authors use a mouse model to show that influenza infection during pregnancy can increase susceptibility to secondary infection and alter immune cell function in offspring.