Differential role of cytosolic Hsp70s in longevity assurance and protein quality control.

70 kDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe reduction in cellular reproduction and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degradation. In contrast, Ssa4 overproduction in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-associated aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overproduction extended the short lifespan of ssa1Δ ssa2Δ mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast.

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

Direct interaction between cholera toxin and dendritic cells is required for oral adjuvant activity.

Cholera toxin (CT) binds to GM1-ganglioside receptors present on all nucleated cells. Despite this, it is a very potent mucosal adjuvant that has a dramatic impact on immune cells, as well as nerve and epithelial cells, causing diarrhea. This fact has hampered our understanding of whether the adjuvanticity of CT is direct or indirect, as cells that bind CT may or may not be involved in its adjuvant function. The mucosal barrier is maintained by tight junctions between epithelial cells but dendritic cells (DCs) can protrude luminal dendrites. Here we investigated which cells are involved in the immune augmenting effect of CT. We explored oral immunizations with ovalbumin (OVA) and CT in bone marrow chimeric mice deficient in GM1-ganglioside in defined cellular subsets. We found that chimeric mice lacking GM1 in nonhematopoietic cells, including epithelial cells, mounted an unaltered intestinal IgA response. In contrast, chimeric mice lacking GM1-expressing hematopoietic cells in general, or specifically GM1-expressing conventional DCs (cDCs), largely failed to elicit anti-OVA adaptive immune responses. Therefore, the adjuvanticity of CT does not require epithelial activation, but is directly dependent on the binding of CT to gut cDCs via GM1-ganglioside. These results could have important implications for the generation of novel oral adjuvants.

The subcellular location of antigen expressed by adenoviral vectors modifies adaptive immunity but not dependency on cross-presenting dendritic cells.

Adenoviral (Ad) vaccine vectors can generate protective immunity to various pathogens in animal studies. However, recent failures in clinical vaccine trials have underscored the need for a better understanding of how mucosal immune responses to Ad-encoded vaccine Ags are generated in vivo. In this study, we addressed whether directing Ad-encoded ovalbumin (OVA) to different subcellular compartments influences the generation of OVA-specific acquired immunity and the APCs required following i.n. immunization of mice. We show that both secreted and membrane-anchored OVA activate CD4(+) T cells, induce cytotoxic CD8(+) T lymphocytes (CTLs) and generate serum IgG. Additionally, vaginal IgG is induced when OVA is expressed at these subcellular locations, but only the secreted form generates a significant IgA response in the lungs. On the contrary, intracellular expression of OVA efficiently expands CD8(+) T cells but fails to activate CD4(+) T cells, results in poor CTL activity, and does not generate Abs. Finally, we show that regardless of the subcellular localization of OVA, conventional DCs (cDCs) are required for the activation of T cells. However, the direct transduction of conventional DCs is not essential. These findings have important implications for the improvement of Ad vector design and vaccine-induced mucosal immunity.

CD11c(high )dendritic cells are essential for activation of CD4+ T cells and generation of specific antibodies following mucosal immunization.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11c(high) conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8(+) T cells in vivo, but the role of cDCs in CD4(+) T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4(+) T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11c(high) cDCs. Our results show that cDCs are absolutely required for activation of CD4(+) T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4(+) T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.