Liquid-Liquid Phase Separation Prediction of Proteins in Salt Solution by Deep Neural Network.

Liquid-liquid phase separation (LLPS) underlies the formation of membrane-free organelles in eukaryotic cells and plays an important role in the development of some diseases. The phase boundary of metastable liquid-liquid phase separation as well as the cloud point temperature of some globular proteins characterize the phase behavior of proteins and have been widely studied theoretically and experimentally. In the present study, we used a regression and classification neural network to deal with the phase behavior of lysozyme and bovine serum albumin (BSA). We predicted the cloud point temperature and solubility of a lysozyme solution containing sodium chloride by regression and the reentrant phase behavior of BSA in YCl3 solution containing a surfactant dodecyl dimethyl amine oxide (DDAO) by classification. Specifically, our network model is capable of predicting (a) the solubility of lysozyme in the range: pH 4.0-5.4, temperature 0-25 °C, and NaCl concentration 2-7% (w/v); (b) the cloud point temperature of lysozyme in the range: pH 4.0-4.8, NaCl concentration 2-7%, and lysozyme concentration 0-400 mg/mL; and (c) the phase behavior of BSA in the range: DDAO 1-60 mM, BSA 30-100 mg/mL, and YCl3 1-20 mM. We experimentally tested the model at some prediction points with a high accuracy, which means that deep neural networks can be applicable in qualitative and quantitive analysis of liquid-liquid phase separation.

The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation.

The CSF-1 receptor (CSF-1R) regulates CNS microglial development. However, the localization and developmental roles of this receptor and its ligands, IL-34 and CSF-1, in the brain are poorly understood. Here we show that compared to wild type mice, CSF-1R-deficient (Csf1r-/-) mice have smaller brains of greater mass. They further exhibit an expansion of lateral ventricle size, an atrophy of the olfactory bulb and a failure of midline crossing of callosal axons. In brain, IL-34 exhibited a broader regional expression than CSF-1, mostly without overlap. Expression of IL-34, CSF-1 and the CSF-1R were maximal during early postnatal development. However, in contrast to the expression of its ligands, CSF-1R expression was very low in adult brain. Postnatal neocortical expression showed that CSF-1 was expressed in layer VI, whereas IL-34 was expressed in the meninges and layers II-V. The broader expression of IL-34 is consistent with its previously implicated role in microglial development. The differential expression of CSF-1R ligands, with respect to CSF-1R expression, could reflect their CSF-1R-independent signaling. Csf1r-/- mice displayed increased proliferation and apoptosis of neocortical progenitors and reduced differentiation of specific excitatory neuronal subtypes. Indeed, addition of CSF-1 or IL-34 to microglia-free, CSF-1R-expressing dorsal forebrain clonal cultures, suppressed progenitor self-renewal and enhanced neuronal differentiation. Consistent with a neural developmental role for the CSF-1R, ablation of the Csf1r gene in Nestin-positive neural progenitors led to a smaller brain size, an expanded neural progenitor pool and elevated cellular apoptosis in cortical forebrain. Thus our results also indicate novel roles for the CSF-1R in the regulation of corticogenesis.

Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells.

CSF-1 is broadly expressed and regulates macrophage and osteoclast development. The action and expression of IL-34, a novel CSF-1R ligand, were investigated in the mouse. As expected, huIL-34 stimulated macrophage proliferation via the huCSF-1R, equivalently to huCSF-1, but was much less active at stimulating mouse macrophage proliferation than huCSF-1. Like muCSF-1, muIL-34 and a muIL-34 isoform lacking Q81 stimulated mouse macrophage proliferation, CSF-1R tyrosine phosphorylation, and signaling and synergized with other cytokines to generate macrophages and osteoclasts from cultured progenitors. However, they respectively possessed twofold and fivefold lower affinities for the CSF-1R and correspondingly, lower activities than muCSF-1. Furthermore, muIL-34, when transgenically expressed in a CSF-1-dependent manner in vivo, rescued the bone, osteoclast, tissue macrophage, and fertility defects of Csf1(op)/(op) mice, suggesting similar regulation of CSF-1R-expressing cells by IL-34 and CSF-1. Whole-mount IL34 in situ hybridization and CSF-1 reporter expression revealed that IL34 mRNA was strongly expressed in the embryonic brain at E11.5, prior to the expression of Csf1 mRNA. QRT-PCR revealed that compared with Csf1 mRNA, IL34 mRNA levels were lower in pregnant uterus and in cultured osteoblasts, higher in most regions of the brain and heart, and not compensatorily increased in Csf1(op/op) mouse tissues. Thus, the different spatiotemporal expression of IL-34 and CSF-1 allows for complementary activation of the CSF-1R in developing and adult tissues.

The propeptide precursor proSAAS is involved in fetal neuropeptide processing and body weight regulation.

Mice with a targeted mutation in proSAAS have been generated to investigate whether peptides derived from this precursor could function as an inhibitor of prohormone convertase 1/3 (PC1/3) in vivo as well as to determine any alternate roles for proSAAS in nervous and endocrine tissues. Fetal mice lacking proSAAS exhibit complete, adult-like processing of prodynorphin in the prenatal brain instead of the incomplete processing seen in the brains of wild-type fetal mice where inhibitory proSAAS intermediates are transiently accumulated. This study provides evidence that proSAAS is directly involved in the prenatal regulation of neuropeptide processing in vivo. However, adult mice lacking proSAAS have normal levels of all peptides detected using a peptidomics approach, suggesting that PC1/3 activity is not affected by the absence of proSAAS in adult mice. ProSAAS knockout mice exhibit decreased locomotion and a male-specific 10-15% decrease in body weight, but maintain normal fasting blood glucose levels and are able to efficiently clear glucose from the blood in response to a glucose challenge. This work suggests that proSAAS-derived peptides can inhibit PC1/3 in embryonic brain, but in the adult brain proSAAS peptides may function as neuropeptides that regulate body weight and potentially other behaviors.

Transgenic expression of CSF-1 in CSF-1 receptor-expressing cells leads to macrophage activation, osteoporosis, and early death.

CSF-1 is the primary mononuclear phagocyte and osteoclast growth factor. Autocrine regulation by CSF-1 has been reported in macrophages during inflammatory responses and in neoplastic cells. To investigate whether inflammatory disease or neoplasia was the dominant consequence of autocrine regulation by CSF-1 in CSF-1 receptor (CSF-1R)-expressing cells, we created mice that express CSF-1 under the control of the CSF-1R promoter/first intron driver [transgene TgN(Csf1r-Csf1)Ers (TgRC) mice], which have reduced thymic size, a short lifetime, and low body weight and develop osteoporosis. In 4-week-old TgRC mice, osteoclast numbers are elevated, and macrophage densities are increased in bone marrow, spleen, liver, and brain. Cultured TgRC macrophages express CSF-1 and proliferate without exogenous CSF-1 and in the presence of neutralizing antimouse CSF-1 antibody. Compared with macrophages from nontransgenic littermates, TgRC macrophages exhibit a stellate morphology, express elevated mRNAs for proinflammatory cytokines, and despite a lower, steady-state cytokine secretion, secrete elevated levels of inflammatory cytokines in response to LPS, indicating that TgRC macrophages are functionally primed through the CSF-1R. Thus, autocrine regulation of CSF-1R-expressing cells by CSF-1 leads to a severe phenotype that emphasizes the importance of the known, local production of CSF-1 in inflammatory disease.

Modulation of CSF-1-regulated post-natal development with anti-CSF-1 antibody.

Colony-stimulating factor-1 (CSF-1) regulates the survival, proliferation and differentiation of macrophages. CSF-1-deficient mice are osteopetrotic due to a lack of osteoclasts, while their tissue macrophage deficiencies and an absence of CSF-1 regulation of CSF-1 receptor-expressing cells in the female reproductive tract contribute to their pleiotropic phenotype. To further understand CSF-1 regulation of macrophages in vivo, we developed a neutralizing anti-mouse CSF-1 antibody which was expressed as a recombinant Fab' fragment and coupled to 40 kDa polyethylene glycol. As developmental regulation by CSF-1 is highest during the early post-natal period, the ability of this anti-CSF-1 reagent to inhibit development was tested by regular subcutaneous injection of mice from post-natal days 0.5-57.5. Antibody treatment decreased growth rate, decreased osteoclast number, induced osteopetrosis, decreased macrophage density in bone marrow, liver, dermis, synovium and kidney and decreased adipocyte size in adipose tissue, thereby inducing phenotypes shared by CSF-1- and CSF-1 receptor-deficient mice. While the antibody blocked macrophage development in some tissues, macrophage densities in other tissues were initially high and were reduced by treatment, proving that the antibody also blocked macrophage maintenance. Since cell surface CSF-1 is sufficient for the maintenance of normal synovial macrophage densities, these studies suggest that anti-CSF-1 Fab'-PEG efficiently neutralizes all three CSF-1 isoforms in vivo, namely the secreted proteoglycan, secreted glycoprotein and cell surface glycoprotein. Since CSF-1 has been shown to enhance chronic disease development in a number of mouse model systems, these studies demonstrate the feasibility of neutralizing CSF-1 effects in these models with an anti-CSF-1 antibody.

Obesity and diabetes in transgenic mice expressing proSAAS.

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.

Neuropeptide-processing carboxypeptidases.

Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this "traditional" pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named "CPA-5," has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites.

Identification and characterization of three members of the human metallocarboxypeptidase gene family.

Amino acid homology searches of the human genome revealed three members of the metallocarboxypeptidase (metallo-CP) family that had not been described in the literature in addition to the 14 known genes. One of these three, named CPA5, is present in a gene cluster with CPA1, CPA2, and CPA4 on chromosome 7. The cDNA encoding a mouse homolog of human CPA5 was isolated from a testis library and sequenced. The deduced amino acid sequence of human CPA5 has highest amino acid sequence identity (60%) to CPA1. Modeling analysis shows the overall structure to be very similar to that of other members of the A/B subfamily of metallocarboxypeptidases. The active site of CPA5 is predicted to cleave substrates with C-terminal hydrophobic residues, as do CPA1, -2, and -3. Using Northern blot analysis, CPA5 mRNA is detected in testis but not in kidney, liver, brain, or lung. In situ hybridization analysis shows that CPA5 is localized to testis germ cells. Mouse pro-CPA5 protein expressed in Sf9 cells using the baculovirus system was retained in the particulate fraction of the cells and was not secreted into the media. Pro-CPA5 was not enzymatically active toward standard CPA substrates, but after incubation with prohormone convertase 4 the resulting protein was able to cleave furylacryloyl-Gly-Leu, with 3-4-fold greater activity at pH 7.4 than at 5.6. Two additional members of the human CP gene family were also studied. Modeling analysis indicates that both contain the necessary amino acids required for enzymatic activity. The CP on chromosome 8 is predicted to have a CPA-like specificity for C-terminal hydrophobic residues and was named CPA6. The CP on chromosome 2 is predicted to cleave substrates with C-terminal acidic residues and was named CPO.