The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43.

The TAR DNA-binding protein of 43kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43's intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43's functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43's physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320-340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.

Multi-functional norrin is a ligand for the LGR4 receptor.

Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.

Ovarian regulation of neuromedin U and its local actions in the ovary, mediated through neuromedin U receptor 2.

Neuromedin U (NMU) was originally identified as an anorexigenic peptide that modulates appetite as well as energy homeostasis through the brain-gut axis. Although growing evidence has linked NMU activity with the development of female reproductive organs, no direct expression of and function for NMU in these organs has been pinpointed. Using a superovulated rat model, we found that NMU is directly expressed in the ovary, where its transcript level is tightly regulated by gonadotropins. Ovarian microdissection and immunohistochemical staining showed clearly that NMU is expressed mainly in theca/interstitial cells and to a moderate extent in granulosa cells. Primary cell studies together with reporter assays indicated the Nmu mRNA level in these cells is strongly induced via cAMP signaling, whereas this increase in expression can be reversed by the degradation message residing within its 3'-untranslated region, which recruits cis-acting mRNA degradation mechanisms, such as the gonadotropin-induced zinc finger RNA-binding protein Zfp36l1. This study also demonstrated that NMUR2, but not NMUR1, is the dominant NMU receptor in the ovary, where its expression is restricted to theca/interstitial cells. Treatment with NMU led to induction of the early response c-Fos gene, phosphorylation of extracellular signal-regulated kinase 1/2, and promotion of progesterone production in both developing and mature theca/interstitial cells. Taken as a whole, this study demonstrates that NMU and NMU receptor 2 compose a novel autocrine system in theca/interstitial cells in which the intensity of signaling is tightly controlled by gonadotropins.

CXCL17, an orphan chemokine, acts as a novel angiogenic and anti-inflammatory factor.

Chemokines play pivotal roles in the recruitment of various immune cells to diverse tissues in both physiological and pathological conditions. CXCL17 is an orphan chemokine preliminarily found to be involved in tumor angiogenesis. However, its protein nature, as well as its endogenous bioactivity, has not been well clarified. Using real-time PCR, immunohistochemical staining, and Western blotting, we found that CXCL17 is highly expressed in both a constitutive and inducible manner in the rat gastric mucosa, where it undergoes endoproteolysis during protein maturation. The mature CXCL17 exhibited strong chemoattractant abilities targeting monocytes and macrophages, potentially through ERK1/2 and p38 but not JNK signaling. CXCL17 also induced the production of proangiogenic factors such as vascular endothelial growth factor A from treated monocytes. Furthermore, in contrast to other CXC chemokines that accelerate inflammatory responses, CXCL17 showed novel anti-inflammatory effects on LPS-activated macrophages. Therefore, our data suggest that CXCL17 in the gastric lamina propria may play an important role in tissue repair and anti-inflammation, both of which help to maintain the integrity of the gastric mucosa.

The Nearest-Neighbor Effect on Random-Coil NMR Chemical Shifts Demonstrated Using a Low-Complexity Amino-Acid Sequence.

In NMR experiments, the chemical shift is typically the first parameter measured and is a source of structural information for biomolecules. Indeed, secondary chemical shifts, the difference between the measured chemical shifts and those expected for a randomly oriented sequence of peptides (the "random coil"), are correlated with the secondary structure of proteins; secondary shift analysis is thereby a standard approach in structural biology. For intrinsically disordered or denatured proteins furthermore, secondary chemical shifts reveal the propensity of particular segments to form different secondary structures. However, because the atoms in unfolded proteins all have very similar chemical environments, the chemical shifts measured for a certain atom type vary less than in globular proteins. Since chemical shifts can be measured precisely, the secondary chemical shifts calculated for an unfolded system depend mainly on the particular random coil chemical shift database chosen as a point of reference. Certain databases correct the random coil shift for a given residue based on its neighbors in the amino acid sequence. However, these corrections are typically derived from the analysis of model peptides; there have been relatively few direct and systematic studies of the effect of neighboring residues for specific amino acid sequences in disordered proteins. For the study reported here, we used the intrinsically disordered C-terminal domain of TDP-43, which has a highly repetitive amino-acid sequence, as a model system. We assigned the chemical shifts of this protein at low pH in urea. Our results demonstrate that the identity of the nearest neighbors is decisive in determining the value of the chemical shift for atoms in a random coil arrangement. Based on these observations, we also outline a possible approach to construct a random-coil library of chemical shifts that comprises all possible arrangement of tripeptides from a manageable number of polypeptides.

A naturally occurring Lgr4 splice variant encodes a soluble antagonist useful for demonstrating the gonadal roles of Lgr4 in mammals.

Leucine-rich repeat containing G protein-coupled receptor 4 (LGR4) promotes the Wnt signaling through interaction with R-spondins or norrin. Using PCR amplification from rat ovarian cDNAs, we identified a naturally occurring Lgr4 splice variant encoding only the ectodomain of Lgr4, which was named Lgr4-ED. Lgr4-ED can be detected as a secreted protein in the extracts from rodent and bovine postnatal gonads, suggesting conservation of Lgr4-ED in mammals. Recombinant Lgr4-ED purified from the conditioned media of transfected 293T cells was found to dose-dependently inhibit the LGR4-mediated Wnt signaling induced by RSPO2 or norrin, suggesting that it is capable of ligand absorption and could have a potential role as an antagonist. Intraperitoneal injection of purified recombinant Lgr4-ED into newborn mice was found to significantly decrease the testicular expression of estrogen receptor alpha and aquaporin 1, which is similar to the phenotype found in Lgr4-null mice. Administration of recombinant Lgr4-ED to superovulated female rats can also decrease the expression of estrogen receptor alpha, aquaporin 1, LH receptor and other key steroidogenic genes as well as bring about the suppression of progesterone production. Thus, these findings suggest that endogenously expressed Lgr4-ED may act as an antagonist molecule and help to fine-tune the R-spondin/norrin-mediated Lgr4-Wnt signaling during gonadal development.