Erratum: Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

This corrects the article DOI: 10.1038/nature24042.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

When the Scaffold Cannot Be Ignored: The Role of the Hydrophobic Core in Ligand Binding and Specificity.

The traditional view of protein-ligand binding treats a protein as comprising distinct binding epitopes on the surface of a degenerate structural scaffold, largely ignoring the impact of a protein's energy landscape. To determine the robustness of this simplification, we compared two small helix-turn-helix transcription factors with different energy landscapes. λ-Repressor is stable and well folded, while MarA appears to be marginally stable with multiple native conformations (molten). While λ-repressor is known to tolerate any hydrophobic mutation in the core, we find MarA drastically less tolerant to core mutation. Moreover, core mutations in MarA (distant from the DNA-binding interface) change the relative affinities of its binding partners, altering ligand specificity. These results can be explained by taking into account the effects of mutations on the entire energy landscape and not just the native state. Thus, for proteins with multiple conformations that are close in energy, such as many intrinsically disordered proteins, residues distant from the active site can alter both binding affinity and specificity.

A single mutation at residue 25 populates the folding intermediate of E. coli RNase H and reveals a highly dynamic partially folded ensemble.

Understanding the nature of partially folded intermediates transiently populated during protein folding is important for understanding both protein folding and misfolding. These ephemeral species, however, often elude direct experimental characterization. The well-characterized protein ribonuclease H (RNase H) from Escherichia coli populates an on-pathway intermediate identified in both bulk studies and single-molecule mechanical unfolding experiments. Here, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. Surprisingly, a single change at Ile25 (I25A) resulted in the equilibrium population of the intermediate under near-native conditions. The intermediate was undetectable in a series of heteronuclear single quantum coherences, revealing the dynamic nature of this partially unfolded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that they are well packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding.

Analytical and statistical methods to evaluate microsatellite allelic imbalance in small amounts of DNA.

Microsatellite analysis is a powerful tool for the assessment of genetic instability and loss of heterozygosity in cancer cells. However, most human tumors harbor significant numbers of normal cells, which may contribute to false-negative results. Recent techniques based on fluorescently labeled primers and semiautomated capillary electrophoresis of polymerase chain reaction (PCR) products allow a reliable quantitative assessment of (PCR) products while requiring very small numbers of cells. We report a highly sensitive protocol for the semiautomated analysis of allelic imbalance based on time-release PCR and capillary electrophoresis. With this protocol, as few as 100 cells can be used to reliably assess allelic imbalance (AI) in DNA samples. Using a panel of seven microsatellite markers, we determined allelic variation in a large set of heterozygous lymphocyte DNA samples and examined the use of different statistical analysis techniques. Using these statistical approaches, we describe a calibration method to evaluate AI from microsatellite results. Using a simple formula, cutoff points at preset confidence levels are used to decide whether allelic imbalance exists in a given sample at the loci under investigation. Our method allows the reliable detection of AI with very small amounts of DNA, and is sufficiently quantitative to assess allelic ratios in nonclonal tissue specimens.