Smaug1 membrane-less organelles respond to AMPK and mTOR and affect mitochondrial function.

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections.

BACKGROUND: The Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a 'mosaic' structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response. METHODS/PRINCIPLE FINDINGS: High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection. CONCLUSIONS/SIGNIFICANCE: In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection.