Abnormal regulation of TSG101 in mice with spongiform neurodegeneration

Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a “PSAP” motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.     

Levels of the Mahogunin Ring Finger 1 E3 Ubiquitin Ligase Do Not Influence Prion Disease

Prion diseases are rare but invariably fatal neurodegenerative disorders. They are associated with spongiform encephalopathy, a histopathology characterized by the presence of large, membrane-bound vacuolar structures in the neuropil of the brain. While the primary cause is recognized as conversion of the normal form of prion protein (PrP^C) to a conformationally distinct, pathogenic form (PrP^Sc), the cellular pathways and mechanisms that lead to spongiform change, neuronal dysfunction and death are not known. Mice lacking the Mahogunin Ring Finger 1 (MGRN1) E3 ubiquitin ligase develop spongiform encephalopathy by 9 months of age but do not become ill. In cell culture, PrP aberrantly present in the cytosol was reported to interact with and sequester MGRN1. This caused endo-lysosomal trafficking defects similar to those observed when Mgrn1 expression is knocked down, implicating disrupted MGRN1-dependent trafficking in the pathogenesis of prion disease. As these defects were rescued by over-expression of MGRN1, we investigated whether reduced or elevated Mgrn1 expression influences the onset, progression or pathology of disease in mice inoculated with PrP^Sc. No differences were observed, indicating that disruption of MGRN1-dependent pathways does not play a significant role in the pathogenesis of transmissible spongiform encephalopathy.     

Transgenic analysis of the physiological functions of Mahogunin ring finger‐1 isoforms

Mahogunin Ring Finger‐1 (Mgrn1) null mutant mice have a pleiotropic phenotype that includes the absence of yellow hair pigment, abnormal head shape, reduced viability, and adult‐onset spongiform neurodegeneration. Mgrn1 encodes a highly conserved E3 ubiquitin ligase with four different isoforms which are differentially expressed and predicted to localize to different subcellular compartments. To test whether loss of specific isoforms causes different aspects of the mutant phenotype, we generated transgenes for each isoform and bred them onto the null mutant background. Mice expressing only isoform I or III appeared completely normal. Isoform II rescued or partially rescued the mutant phenotypes, whereas isoform IV had little or no effect. Our data show that different Mgrn1 isoforms are not functionally equivalent in vivo and that the presence of only isoform I or III is sufficient for normal development, pigmentation, and neuronal integrity. genesis 47:524–534, 2009. © 2009 Wiley‐Liss, Inc.     

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule dynamics and intracellular transport

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1‐mediated ubiquitination of α‐tubulin in interphase cells. Here, we show that MGRN1‐mediated ubiquitination regulates dynamics of EB1‐labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule‐dependent organellar transport are evident in cells where noncanonical K6‐mediated ubiquitination of α‐tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late‐onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (^CtmPrP) interacts with MGRN1 leading to its loss of function. Expression of ^CtmPrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1‐mediated ubiquitination of α‐tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non‐transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.      

The mahoganoid mutation (Mgrn1md) improves insulin sensitivity in mice with mutations in the melanocortin signaling pathway independently of effects on adiposity

Mahoganoid (Mgrn1(md)) is a mutation of the mahogunin (Mgrn1) gene. The hypomorphic allele suppresses the yellow pigmentation and obesity of the A(y) mouse that ubiquitously overexpresses agouti signaling protein (ASP). To assess the physiological effects of MGRN1 on energy and glucose homeostasis, we generated animals doubly mutant for Mgrn1(md) and A(y), Lep(ob), or a null allele of Mc4r, and diet-induced obesity (DIO) mice segregating for Mgrn1(md). Mgrn1(md) suppressed the obesity, hyperglycemia, and hyperinsulinemia of A(y) mice. Mgrn1(md) suppressed A(y)-induced obesity by reducing food intake, and reduced adiposity in Lep(ob)/Lep(ob) females, but did not alter the body weight or body composition of mice fed a high-fat diet. There was no effect of Mgrn1(md) on weight gain, body composition, energy intake, or energy expenditure in Mc4r-null animals. Mgrn1(md) reduced circulating insulin concentrations in DIO, A(y), and Mc4r-null but not Lep(ob)/Lep(ob) mice. The effect of Mgrn1(md) on circulating insulin concentrations was not due primarily to reductions in fat mass, since the plasma insulin concentrations of Mgrn1(md) mice segregating for either A(y) or Mc4r-null alleles, adjusted for fat mass and plasma glucose, were reduced compared with A(y) and Mc4r mice, respectively. The effect of Mgrn1(md) on insulin sensitivity of Mc4r-null mice suggests that Mgrn1(md) may be increasing insulin sensitivity via the hypothalamic melanocortin-3 receptor pathway.     

Mahogunin-mediated α-tubulin ubiquitination via noncanonical K6 linkage regulates microtubule stability and mitotic spindle orientation

Mahogunin ring finger-1 (MGRN1) is a cytosolic ubiquitin ligase whose disruption or interaction with some isoforms of cytosolically exposed prion protein leads to spongiform neurodegeneration and also lack of which results in reduced embryonic viability due to mispatterning of the left–right (LR) axis during development. Here we demonstrate an interaction between the cytoskeletal protein α-tubulin and MGRN1. In cultured cell systems, loss of the ubiquitin E3 ligase activity of MGRN1 results in spindle misorientation and decreased α-tubulin polymerization, an effect also seen in primary cells. α-Tubulin was post-translationally modified by MGRN1 via noncanonical K6-linked polyubiquitination. This was significant because expression of catalytically inactive MGRN1 and/or ubiquitin mutant capable of only monoubiquitination resulted in similar mitotic spindle misorientation. The modulatory effect of MGRN1 was specific for α-tubulin and similar changes could not be detected in β- or γ-tubulin. However, catalytic inactivation of MGRN1 did not abrogate monoubiquitination of α-tubulin, thus unraveling a unique dual mode of ubiquitination by an unknown E3 ligase and MGRN1. MGRN1-mediated α-tubulin modification, and hence its stability, may highlight a key event in the LR patterning during embryogenesis.     

Concentration of Disease-Associated Prion Protein with Silicon Dioxide

Reagents that can precipitate the disease-associated prion protein (PrP^Sc) are vital for the development of high sensitivity tests to detect low levels of this disease marker in biological material. Here, a range of minerals are shown to precipitate both ovine cellular prion protein (PrP^C) and ovine scrapie PrP^Sc. The precipitation of prion protein with silicon dioxide is unaffected by PrP^Sc strain or host species and the method can be used to precipitate bovine BSE. This method can reliably concentrate protease-resistant ovine PrP^Sc (PrP^res) derived from 1.69 μg of brain protein from a clinically infected animal diluted into either 50 ml of buffer or 15 ml of plasma. The introduction of a SiO₂ precipitation step into the immunological detection of PrP^res increased detection sensitivity by over 1,500-fold. Minerals such as SiO₂ are readily available, low cost reagents with generic application to the concentration of diseases-associated prion proteins.     

Molecular dynamics studies on the buffalo prion protein

It was reported that buffalo is a low susceptibility species resisting to transmissible spongiform encephalopathies (TSEs) (same as rabbits, horses, and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (except for rabbits, dogs, horses, and buffalo), manifesting as scrapie in sheep and goats; bovine spongiform encephalopathy (BSE or “mad–cow” disease) in cattle; chronic wasting disease in deer and elk; and Creutzfeldt–Jakob diseases, Gerstmann–Sträussler–Scheinker syndrome, fatal familial insomnia, and Kulu in humans etc. In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP^C), predominantly with α-helices, into insoluble abnormally folded infectious prions (PrP^Sc), rich in β-sheets. In this article, we studied the molecular structure and structural dynamics of buffalo PrP^C (BufPrP^C), in order to understand the reason why buffalo is resistant to prion diseases. We first did molecular modeling of a homology structure constructed by one mutation at residue 143 from the NMR structure of bovine and cattle PrP(124–227); immediately we found that for BufPrP^C(124–227), there are five hydrogen bonds (HBs) at Asn143, but at this position, bovine/cattle do not have such HBs. Same as that of rabbits, dogs, or horses, our molecular dynamics studies also revealed there is a strong salt bridge (SB) ASP178–ARG164 (O–N) keeping the β2–α2 loop linked in buffalo. We also found there is a very strong HB SER170–TYR218 linking this loop with the C-terminal end of α-helix H3. Other information, such as (i) there is a very strong SB HIS187–ARG156 (N–O) linking α-helices H2 and H1 (if mutation H187R is made at position 187, then the hydrophobic core of PrP^C will be exposed (L.H. Zhong (2010). Exposure of hydrophobic core in human prion protein pathogenic mutant H187R. Journal of Biomolecular Structure and Dynamics 28(3), 355–361)), (ii) at D178, there is a HB Y169–D178 and a polar contact R164–D178 for BufPrP^C instead of a polar contact Q168–D178 for bovine PrP^C (C.J. Cheng, & V. Daggett. (2014). Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH. Biomolecules 4(1), 181–201), (iii) BufPrP^C owns three 3₁₀ helices at 125–127, 152–156, and in the β2–α2 loop, respectively, and (iv) in the β2–α2 loop, there is a strong π–π stacking and a strong π–cation F175–Y169–R164.(N)NH2, has been discovered.     

Prion protein polymerisation triggered by manganese-generated prion protein seeds

Prion diseases are neurodegenerative diseases that can be transmitted between individuals. The exact cause of these diseases remains unknown. However, one of the key events associates with the disease is the aggregation of a cellular protein, the prion protein. The mechanism of this is still unclear. However, it is likely that the aggregation is trigged by a seeding mechanism in which an oligomer of the prion protein is able to catalyse polymerisation of further prion protein into larger aggregates. We have developed a model of this process using an oligomeric species generated from recombinant protein by exposure to manganese. On fractionation of the seeding species, we estimated that the smallest size the oligomer would be is an octomer. We analysed the catalytic mechanism of the seeding oligomer and its interaction with substrate. Different domains of the protein are necessary for the seeding ability of the prion protein as opposed to those required for it to form a substrate for the polymerisation reaction. Prion seeds formed from different sheep alleles are able to reproduce the characteristics of scrapie in terms of resistance to disease. However, we were also able to generate prion seed from chicken PrP a species where no prion disease is known. Our findings provide an insight into the aggregation process of the prion protein and its potential relation to disease progress.     

Post‐translational changes to PrP alter transmissible spongiform encephalopathy strain properties

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP^Sc, an abnormal conformer of the host glycoprotein PrP^C. TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP^Sc with the same amino‐acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post‐translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild‐type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N‐glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild‐type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain‐specific characteristics of the 79A TSE strain changed when PrP^Sc was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post‐translational changes to PrP which we propose result in the selection of mutant TSE strains.     

Agouti protein,mahogunin, and attractin in pheomelanogenesis and melanoblast‐like alteration of melanocytes: a cAMP‐independent pathway

Melanocortin‐1 receptor (MC1R) and its ligands, α‐melanocyte stimulating hormone (αMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan‐a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200‐fold increases in the pheomelanin to eumelanin ratio, and a tan‐yellow color in pelletted cells. Moreover, ASIP‐treated cells showed reduced proliferation and a melanoblast‐like appearance, seen also in melanocyte lines from yellow (A^y/a and Mc1r^e/ Mc1r^e) mice. However ASIP‐YY, a C‐terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP‐YY inhibited the cAMP rise induced by αMSH analog NDP‐MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrn^{mg‐3J/mg‐3J} or Mgrn1^{md‐nc/md‐nc}) also responded to both ASIP and ASIP‐YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP–MC1R signaling includes a cAMP‐independent pathway through attractin and mahogunin, while the known cAMP‐dependent component requires neither attractin nor mahogunin.     

Mahogunin regulates fusion between amphisomes/MVBs and lysosomes via ubiquitination of TSG101

Aberrant metabolic forms of the prion protein (PrP), membrane-associated ^CtmPrP and cytosolic (cyPrP) interact with the cytosolic ubiquitin E3 ligase, Mahogunin Ring Finger-1 (MGRN1) and affect lysosomes. MGRN1 also interacts with and ubiquitinates TSG101, an ESCRT-I protein, involved in endocytosis. We report that MGRN1 modulates macroautophagy. In cultured cells, functional depletion of MGRN1 or overexpression of ^CtmPrP and cyPrP blocks autophagosome–lysosome fusion, alleviates the autophagic flux and its degradative competence. Concurrently, the degradation of cargo from the endo-lysosomal pathway is also affected. This is significant because catalytic inactivation of MGRN1 alleviates fusion of lysosomes with either autophagosomes (via amphisomes) or late endosomes (either direct or mediated through amphisomes), without drastically perturbing maturation of late endosomes, generation of amphisomes or lysosomal proteolytic activity. The compromised lysosomal fusion events are rescued by overexpression of TSG101 and/or its monoubiquitination in the presence of MGRN1. Thus, for the first time we elucidate that MGRN1 simultaneously modulates both autophagy and heterophagy via ubiquitin-mediated post-translational modification of TSG101.All cells rely on efficient lysosomal degradation for maintenance of their homoeostasis, perturbations in this leads to several debilitating diseases. Lysosomes are specialized organelles that degrade macromolecules received from the secretory, endocytic, autophagic and phagocytic pathways. Autophagy is considered as a ubiquitous bulk degradation mechanism of damaged organelles and long lived, misfolded or accumulated proteins.¹ Activated growth factors, hormones, cytokine receptors, misfolded plasma membrane proteins are internalized by endocytosis and delivered to the lysosomes via the multivesicular bodies (MVBs), a mechanism also termed as heterophagy. Interestingly defects in either of the pathways have been associated with the pathogenesis of numerous neurodegenerative diseases.²Perturbations in autophagy-related protein (ATG) genes, Atg7 and Atg5 lead to developmental defects during organogenesis^{3, 4} or even neonatal death.⁵ Similarly, studies have reported that null mutations in the lysosomal membrane protein LAMP2 result in general myopathy and cardiomyopathy.^{6, 7} Lysosomal degradation is essential for normal physiological activity in neurons. Anomalies at various stages in the maturation of the endosomes through MVBs to lysosomes or during the de novo generation of autophagosomes result in neurodegenerative diseases like Alzheimer''s disease and Huntington''s disease.^{8, 9}Many other neurodegenerative diseases like Parkinson''s disease, Niemann–Pick type C disease, frontotemporal dementia (FTD) and amyotropic lateral sclerois (ALS) are also referred as ‘lysosomal diseases''. These are all associated with dysfunction of the ESCRT (endosomal sorting complex required for transport) machinery, comprising a pathway of five distinct complexes (ESCRTs -0, -I, -II and -III, and Vps4), which recognize and sort ubiquitinated cargo through an exquisite division of labor.¹⁰ Depletion or mutations in the molecular players of the ESCRT complexes severely affects the structure and function of endo-lysosomal compartments.^{11, 12, 13, 14} These proteins also facilitate autophagy by affecting fusion events involving lysosomes, endosomes and autophagosomes.^{15, 16, 17, 18, 19, 20}In context of this, it is worth indicating that loss of Mgrn1 (Mahogunin Ring Finger-1) function leads to late-onset spongiform neurodegeneration in selected brain regions, very similar to prion disease pathology.²¹ Catalytically MGRN1, a cytosolic ubiquitin E3 ligase is implicated in lysosomal dysfunction.^{22, 23} MGRN1 can interact with a transmembrane prion protein (PrP) isoform (^CtmPrP), associated with familial or inherited disease.²³ It is also suggested to be involved in the clearance of cytosolic chaperone heat shock 70 kDa protein (HSP70)-associated misfolded proteins.²⁴ Although it is prudent to suggest that MGRN1 could have a role in certain familial prion diseases, recent evidence does not indicate its involvement in transmissible spongiform encephalopathy.²⁵ However, this does not undermine the role of MGRN1 in regulating lysosomal degradation.Here, we dissect the mechanism by which MGRN1 regulates lysosomal degradation. We have identified a novel role MGRN1 in modulating autophagy. Depletion of MGRN1 disrupts both amphisomal–lysosomal and endo-lysosomal degradation pathways. These effects are due to the blocked fusion of vesicles with lysosomes and can be rescued by overexpression of TSG101 and/or its monoubiquitination. MGRN1 can modulate clearance of cargo at the lysosomes by regulating vesicular fusion events.     

Genetic analysis of attractin homologs

Attractin (ATRN) and Attractin-like 1 (ATRNL1) are highly similar type I transmembrane proteins. Atrn null mutant mice have a pleiotropic phenotype including dark fur, juvenile-onset spongiform neurodegeneration, hypomyelination, tremor, and reduced body weight and adiposity, implicating ATRN in numerous biological processes. Bioinformatic analysis indicated that Atrn and Atrnl1 arose from a common ancestral gene early in vertebrate evolution. To investigate the genetics of the ATRN system and explore potential redundancy between Atrn and Atrnl1, we generated and characterized Atrnl1 loss- and gain-of-function mutations in mice. Atrnl1 mutant mice were grossly normal with no alterations of pigmentation, central nervous system pathology or body weight. Atrn null mutant mice carrying a beta-actin promoter-driven Atrnl1 transgene had normal, agouti-banded hairs and significantly delayed onset of spongiform neurodegeneration, indicating that over-expression of ATRNL1 compensates for loss of ATRN. Thus, the two genes are redundant from the perspective of gain-of-function but not loss-of-function mutations.     

Transgenic Fatal Familial Insomnia Mice Indicate Prion Infectivity-Independent Mechanisms of Pathogenesis and Phenotypic Expression of Disease

Fatal familial insomnia (FFI) and a genetic form of Creutzfeldt-Jakob disease (CJD¹⁷⁸) are clinically different prion disorders linked to the D178N prion protein (PrP) mutation. The disease phenotype is determined by the 129 M/V polymorphism on the mutant allele, which is thought to influence D178N PrP misfolding, leading to the formation of distinctive prion strains with specific neurotoxic properties. However, the mechanism by which misfolded variants of mutant PrP cause different diseases is not known. We generated transgenic (Tg) mice expressing the mouse PrP homolog of the FFI mutation. These mice synthesize a misfolded form of mutant PrP in their brains and develop a neurological illness with severe sleep disruption, highly reminiscent of FFI and different from that of analogously generated Tg(CJD) mice modeling CJD¹⁷⁸. No prion infectivity was detectable in Tg(FFI) and Tg(CJD) brains by bioassay or protein misfolding cyclic amplification, indicating that mutant PrP has disease-encoding properties that do not depend on its ability to propagate its misfolded conformation. Tg(FFI) and Tg(CJD) neurons have different patterns of intracellular PrP accumulation associated with distinct morphological abnormalities of the endoplasmic reticulum and Golgi, suggesting that mutation-specific alterations of secretory transport may contribute to the disease phenotype.     

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.     

Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G��s

Mahogunin ring finger-1 (MGRN1) is a RING domain-containing ubiquitin ligase mutated in mahoganoid, a mouse mutation causing coat color darkening, congenital heart defects, high embryonic lethality, and spongiform neurodegeneration. The melanocortin hormones regulate pigmentation, cortisol production, food intake, and body weight by signaling through five G protein-coupled receptors positively coupled to the cAMP pathway (MC1R–MC5R). Genetic analysis has shown that mouse Mgrn1 is an accessory protein for melanocortin signaling that may inhibit MC1R and MC4R by unknown mechanisms. These melanocortin receptors (MCRs) regulate pigmentation and body weight, respectively. We show that human melanoma cells express 4 MGRN1 isoforms differing in the C-terminal exon 17 and in usage of exon 12. This exon contains nuclear localization signals. MGRN1 isoforms decreased MC1R and MC4R signaling to cAMP, without effect on β₂-adrenergic receptor. Inhibition was independent on receptor plasma membrane expression, ubiquitylation, internalization, or stability and occurred upstream of Gα_s binding to/activation of adenylyl cyclase. MGRN1 co-immunoprecipitated with MCRs, suggesting a physical interaction of the proteins. Significantly, overexpression of Gα_s abolished the inhibitory effect of MGRN1 and decreased co-immunoprecipitation with MCRs, suggesting competition between MGRN1 and Gα_s for binding to MCRs. Although all MGRN1s were located in the cytosol in the absence of MCRs, exon 12-containing isoforms accumulated in the nuclei upon co-expression with the receptors. Therefore, MGRN1 inhibits MCR signaling by a new mechanism involving displacement of Gα_s, thus accounting for key features of the mahoganoid phenotype. Moreover, MGRN1 might provide a novel pathway for melanocortin signaling from the cell surface to the nucleus.     

In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrP^Sc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrP^Sc from 3 distinct experimental isolates of H-BSE, could detect PrP^Sc in as little as 1×10^?12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrP^Sc with distinct protease K-resistance and protease K-resistant PrP^Sc molecular weights.