RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

Human α2-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98-132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct β-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new "bilobal" or the classic "standard" mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system.

Dysregulation of the complement system is central to age-related macular degeneration (AMD), the leading cause of blindness in the developed world. Most of the genetic variation associated with AMD resides in complement genes, with the greatest risk associated with polymorphisms in the complement factor H (CFH) gene; factor H (FH) is the major inhibitor of the alternative pathway (AP) of complement that specifically targets C3b and the AP C3 convertase. Long pentraxin 3 (PTX3) is a soluble pattern recognition molecule that has been proposed to inhibit AP activation via recruitment of FH. Although present in the human retina, if and how PTX3 plays a role in AMD is still unclear. In this work we demonstrated the presence of PTX3 in the human vitreous and studied the PTX3-FH-C3b crosstalk and its effects on complement activation in a model of retinal pigment epithelium (RPE). RPE cells cultured in inflammatory AMD-like conditions overexpressed the PTX3 protein, and up-regulated AP activating genes. PTX3 bound RPE cells in a physiological setting, however this interaction was reduced in inflammatory conditions, whereby PTX3 had no complement-inhibiting activity on inflamed RPE. However, on non-cellular surfaces, PTX3 formed a stable ternary complex with FH and C3b that acted as a "hot spot" for complement inhibition. Our findings suggest a protective role for PTX3 in response to complement dysregulation in AMD and point to a novel mechanism of complement regulation by this pentraxin with potential implications in pathology and pharmacology of AMD.

Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Potato juice, a by-product of starch processing, is a potential high-value food ingredient due to its high protein content. However, conversion from feed to human protein requires the removal of the toxic antinutritional glycoalkaloids (GAs) α-chaconine and α-solanine. Detoxification by enzymatic removal could potentially provide an effective and environmentally friendly process for potato-derived food protein production. While degradation of GAs by microorganisms has been documented, there exists limited knowledge on the enzymes involved and in particular how bacteria degrade and metabolize GAs. Here we describe a series of methods for the isolation, screening, and selection of GA-degrading bacteria. Bacterial cultures from soils surrounding greened potatoes, including the potato peels, were established and select bacterial isolates were studied. Screening of bacterial crude extracts for the ability to hydrolyze GAs was performed using a combination of thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and liquid chromatography mass spectrometry (LC-MS). Analysis of the 16S rRNA sequences revealed that bacteria within the genus Arthrobacter were among the most efficient GA-degrading strains.

Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD.

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Recent genome-wide association studies (GWAS) have revealed strong association of hypercholesterolemia and myocardial infarction with SNPs on human chromosome 1p13.3. This locus covers three genes: SORT1, CELSR2, and PSRC1. We demonstrate that sortilin, encoded by SORT1, is an intracellular sorting receptor for apolipoprotein (apo) B100. It interacts with apoB100 in the Golgi and facilitates the formation and hepatic export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol. Absence of sortilin in gene-targeted mice reduces secretion of lipoproteins from the liver and ameliorates hypercholesterolemia and atherosclerotic lesion formation in LDL receptor-deficient animals. In contrast, sortilin overexpression stimulates hepatic release of lipoproteins and increases plasma LDL levels. Our data have uncovered a regulatory pathway in hepatic lipoprotein export and suggest a molecular explanation for the cardiovascular risk being associated with 1p13.3.

Proteoglycans (PGs) are essential components of the animal extracellular matrix and are required for cell adhesion, migration, signaling, and immune function. PGs are composed of a core protein and long glycosaminoglycan (GAG) chains, which often specify PG function. GAG biosynthesis is initiated by peptide O-xylosyltransferases, which transfer xylose onto selected serine residues in the core proteins. We have determined crystal structures of human xylosyltransferase 1 (XT1) in complex with the sugar donor, UDP-xylose, and various acceptor peptides. The structures reveal unique active-site features that, in conjunction with functional experiments, explain the substrate specificity of XT1. A constriction within the peptide binding cleft requires the acceptor serine to be followed by glycine or alanine. The remainder of the cleft can accommodate a wide variety of sequences, but with a general preference for acidic residues. These findings provide a framework for understanding the selectivity of GAG attachment.

Renal tubules are highly active transporting epithelia and are at risk of protein aggregation due to high protein turnover and/or oxidative stress. We hypothesized that the risk of aggregation was increased upon hormone stimulation and assessed the state of the intracellular protein degradation systems in the kidney from control rats and rats receiving aldosterone or angiotensin II treatment for 7 days. Control rats formed both aggresomes and autophagosomes specifically in the proximal tubules, indicating a need for these structures even under baseline conditions. Fluorescence sorted aggresomes contained various rat keratins known to be expressed in renal tubules as assessed by protein mass spectrometry. Aldosterone administration increased the abundance of the proximal tubular aggresomal protein keratin 5, the ribosomal protein RPL27, ataxin-3, and the chaperone heat shock protein 70-4 with no apparent change in the aggresome-autophagosome markers. Angiotensin II induced aggregation of RPL27 specifically in proximal tubules, again without apparent change in antiaggregating proteins or the aggresome-autophagosome markers. Albumin endocytosis was unaffected by the hormone administration. Taken together, we find that the renal proximal tubules display aggresome formation and autophagy. Despite an increase in aggregation-prone protein load in these tubules during hormone treatment, renal proximal tubules seem to have sufficient capacity for removing protein aggregates from the cells.

Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE). Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin.

Retinal inflammation plays a key role in the progression of age-related macular degeneration (AMD), a condition that leads to loss of central vision. The deposition of the acute phase pentraxin C-reactive protein (CRP) in the macula activates the complement system, thereby contributing to dysregulated inflammation. The complement protein factor H (FH) can bind CRP and down-regulate an inflammatory response. However, it is not known whether a truncated form of FH, called factor H-like protein 1 (FHL-1), which plays a significant regulatory role in the eye, also interacts with CRP. Here, we compare the binding properties of FHL-1 and FH to both CRP and the related protein pentraxin-3 (PTX3). We find that, unlike FH, FHL-1 can bind pro-inflammatory monomeric CRP (mCRP) as well as the circulating pentameric form. Furthermore, the four-amino acid C-terminal tail of FHL-1 (not present in FH) plays a role in mediating its binding to mCRP. PTX3 was found to be present in the macula of donor eyes and the AMD-associated Y402H polymorphism altered the binding of FHL-1 to PTX3. Our findings reveal that the binding characteristics of FHL-1 differ from those of FH, likely underpinning independent immune regulatory functions in the context of the human retina.

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21(Cip1) in a p53-independent manner, identified the TGFbeta pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated beta-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy.

Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (<1 year) and the child/adult corneas (>6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.

The role of the receptor for advanced glycation end-products (RAGE) has been shown to differ in two different mouse models of asbestos and bleomycin induced pulmonary fibrosis. RAGE knockout (KO) mice get worse fibrosis when challenged with asbestos, whereas in the bleomycin model they are largely protected against fibrosis. In the current study the role of RAGE in a mouse model of silica induced pulmonary fibrosis was investigated.

Exceptional and extreme feeding behaviour makes the Burmese python (Python bivittatus) an interesting model to study physiological remodelling and metabolic adaptation in response to refeeding after prolonged starvation. In this study, we used transcriptome sequencing of 5 visceral organs during fasting as well as 24 hours and 48 hours after ingestion of a large meal to unravel the postprandial changes in Burmese pythons. We first used the pooled data to perform a de novo assembly of the transcriptome and supplemented this with a proteomic survey of enzymes in the plasma and gastric fluid. We constructed a high-quality transcriptome with 34 423 transcripts, of which 19 713 (57%) were annotated. Among highly expressed genes (fragments per kilo base per million sequenced reads > 100 in 1 tissue), we found that the transition from fasting to digestion was associated with differential expression of 43 genes in the heart, 206 genes in the liver, 114 genes in the stomach, 89 genes in the pancreas, and 158 genes in the intestine. We interrogated the function of these genes to test previous hypotheses on the response to feeding. We also used the transcriptome to identify 314 secreted proteins in the gastric fluid of the python. Digestion was associated with an upregulation of genes related to metabolic processes, and translational changes therefore appear to support the postprandial rise in metabolism. We identify stomach-related proteins from a digesting individual and demonstrate that the sensitivity of modern liquid chromatography/tandem mass spectrometry equipment allows the identification of gastric juice proteins that are present during digestion.

Matrix extracellular phosphoglycoprotein (MEPE) is expressed in bone and teeth where it has multiple functions. The C-terminus of MEPE contains a mineral-binding, acidic serine- and aspartate-rich motif (ASARM) that is also present in other noncollagenous proteins of mineralized tissues. MEPE-derived ASARM peptides function in phosphate homeostasis and direct inhibition of bone mineralization in a phosphorylation-dependent manner. MEPE is phosphorylated by family with sequence similarity 20, member C (FAM20C), which is the main kinase phosphorylating secreted phosphoprotein. Although the functional importance of protein phosphorylation status in mineralization processes has now been well-established for secreted bone and tooth proteins (particularly for osteopontin), the phosphorylation pattern of MEPE has not been previously determined. Here we provide evidence for a very high phosphorylation level of this protein, reporting on the localization of 31 phosphoresidues in human MEPE after coexpression with FAM20C in HEK293T cells. This includes the finding that all serine residues located in the canonical target sequence of FAM20C (Ser-x-Glu) were phosphorylated, thus establishing the major target sites for this kinase. We also show that MEPE has numerous other phosphorylation sites, these not being positioned in the canonical phosphorylation sequence. Of note, and underscoring a possible important function in mineralization biology, all nine serine residues in the ASARM were phosphorylated, even though only two of these were positioned in the Ser-x-Glu sequence. The presence of many phosphorylated amino acids in MEPE, and particularly their high density in the ASARM motif, provides an important basis for the understanding of structural and functional interdependencies in mineralization and phosphate homeostasis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.