Goodpasture Antigen-binding Protein Is a Soluble Exportable Protein That Interacts with Type IV Collagen

Goodpasture-antigen binding protein (GPBP) is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Various studies have questioned these findings and proposed that GPBP serves as transporter of ceramide between the endoplasmic reticulum and the Golgi apparatus. Here we show that cells expressed at leasttwoGPBPisoforms resultingfromcanonical (77- kDa) and noncanonical (91-kDa)mRNAtranslation initiation.The 77-kDa polypeptide interacted with type IV collagen and localized as a soluble form in the extracellular compartment. The 91-kDa polypeptide and its derived 120-kDa polypeptide associated with cellular membranes and regulated the extracellular levels of the 77-kDa polypeptide. A short motif containing two phenylalanines in an acidic tract and the 26-residue Ser-rich region were required for efficient 77-kDa polypeptide secretion. Removal of the 26-residue Ser-rich region by alternative exon splicing rendered the protein cytosolic and sensitive to the reduction of sphingomyelin cellular levels. Theseandprevious data implicateGPBPsin a multicompartmental program for protein secretion (i.e. type IV collagen) that includes: 1) phosphorylation and regulation of protein molecular/supramolecular organization and 2) interorganelle ceramide trafficking and regulation of protein cargo transport to the plasma membrane.This work was supported by Grants SAF97/0065, SAF2000/0047, SAF2001/0453, SAF2003-09772-C03-01, and SAF2006-12520-C02-01 from Ministerio de Educacio´ n y Ciencia, Grant 98/102-00 from Fundacio ´ n “La Caixa,” and Grants GV04B-285 and BM-001/2002 from Generalitat Valenciana (Spain) (to J. S.).Medicin

SP1 and NFY Regulate the Expression of PNPT1, a Gene Encoding a Mitochondrial Protein Involved in Cancer

The Polyribonucleotide nucleotidyltransferase 1 gene (PNPT1) encodes polynucleotide phosphorylase (PNPase), a 30-50 exoribonuclease involved in mitochondrial RNA degradation and surveillance and RNA import into the mitochondrion. Here, we have characterized the PNPT1 promoter by in silico analysis, luciferase reporter assays, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), siRNA-based mRNA silencing and RT-qPCR.We show that the Specificity protein 1 (SP1) transcription factor and Nuclear transcription factor Y (NFY) bind the PNPT1 promoter, and have a relevant role regulating the promoter activity, PNPT1 expression, and mitochondrial activity. We also found in Kaplan–Meier survival curves that a high expression of either PNPase, SP1 or NFY subunit A (NFYA) is associated with a poor prognosis in liver cancer. In summary, our results show the relevance of SP1 and NFY in PNPT1 expression, and point to SP1/NFY and PNPase as possible targets in anti-cancer therapy.Medicin

Selective targeting of collagen IV in the cancer cell microenvironment reduces tumor burden

Goodpasture antigen-binding protein (GPBP) is an exportable1 Ser/Thr kinase that induces collagen IV expansion and has been associated with chemoresistance following epithelial-to-mesenchymal transition (EMT). Here we demonstrate that cancer EMT phenotypes secrete GPBP (mesenchymal GPBP) which displays a predominant multimeric oligomerization and directs the formation of previously unrecognized mesh collagen IV networks (mesenchymal collagen IV). Yeast twohybrid (YTH) system was used to identify a 260SHCIE264 motif critical for multimeric GPBP assembly which then facilitated design of a series of potential peptidomimetics. The compound 3-[4''-methoxy-3,2'-dimethyl-(1,1';4',1'')terphenyl-2''-yl]propionic acid, or T12, specifically targets mesenchymal GPBP and disturbs its multimerization without affecting kinase catalytic site. Importantly, T12 reduces growth and metastases of tumors populated by EMT phenotypes. Moreover, low-dose doxorubicin sensitizes epithelial cancer precursor cells to T12, thereby further reducing tumor load. Given that T12 targets the pathogenic mesenchymal GPBP, it does not bind significantly to normal tissues and therapeutic dosing was not associated with toxicity. T12 is a first-in-class drug candidate to treat cancer by selectively targeting the collagen IV of the tumor cell microenvironment.This work was supported by grants: PET 2006_0721, TRA2009_0026, IPT-010000-2010-45, IPT-2011-1527- 010000, RTC-2014-2415-1, PCB-010000-2010-031, PCB- 010000-2010-032, EQU-2014-1-0301 of the Plan Nacional de Investigación, Desarrollo e Innovación of the Spanish Government and IMGESA/06/78, IMGESA/06/79 of Conselleria d’Empresa, Universitat i Ciencia of Generalitat Valenciana to Fibrostatin, S.L. and J.S.; SAF 2001/0453, SAF 2003-09772-C03-01, SAF 2006-12520-C02-01, SAF 2009-10703 of the Plan Nacional de Investigación, Desarrollo e Innovación of the Spanish Government and PROMETEO/2009/065, PROMETEOII/2014/048 of Conselleria de Educaciò of Generalitat Valenciana to J.S. Additional funding came from ERESA, BioStratum Inc. and NephroGenex Inc. R&D programs, and personal funding from Vicente Saus and Carmen Cano to J.S.. Torres Quevedo program of the Spanish Government granted F.R., F-R-R., R.B., E.L-P and A.P-S.Medicin

Unicellular ancestry and mechanisms of diversification of Goodpasture antigen– binding protein

The emergence of the basement membrane (BM), a specialized form of extracellular matrix, was essential in the unicellular transition to multicellularity. However, the mechanism is unknown. Goodpasture antigen– binding protein (GPBP), a BM protein, was uniquely poised to play diverse roles in this transition owing to its multiple isoforms (GPBP-1, -2, and -3) with varied intracellular and extracellular functions (ceramide trafficker and protein kinase). We sought to determine the evolutionary origin of GPBP isoforms. Our findings reveal the presence of GPBP in unicellular protists, with GPBP-2 as the most ancient isoform. In vertebrates, GPBP-1 assumed extracellular function that is further enhanced by membrane-bound GPBP-3 in mammalians, whereas GPBP-2 retained intracellular function. Moreover, GPBP-2 possesses a dual intracellular/extracellular function in cnidarians, an early nonbilaterian group. We conclude that GPBP functioning both inside and outside the cell was of fundamental importance for the evolutionary transition to animal multicellularity and tissue evolution.This work was supported by NIDDK, National Institutes of Health, Grants R01DK 28381-46S1 and R01DK18381; Atracció de Talent from the University of Valencia; El Plan Nacional de Investigación Científica, Desarrollo e Innovación Grant RTC-2014-2415-1; and Conselleria de Educaciò of Generalitat Valenciana Grant PROMETEOII/2014/048; and The Aspirnaut Program (to Julie K. Hudson and B. G. H.).Biotecnologí

More Than Three Years for Normalisation of Routine Laboratory Values after Gluten Withdrawal in Paediatric Coeliac Patients

The assessment of the nutritional and inflammatory status of paediatric patients with coeliac disease is an interesting approach to early diagnosis and functional follow-up. Most authors agree that the normalisation of symptoms takes about one year. The aim of the study was to evaluate the clinical manifestation and normalisation of routine analytics in Spanish children diagnosed with celiac disease. Methods: We performed a retrospective case–control study in Spanish paediatric patients, including 21 celiac patients and 20 healthy controls. The 21 patients selected in the case–control study were followed for 5 years after starting a gluten-free diet (GFD). All patients had type 3 villous atrophy according to the Marsh–Oberhuber classification. A total of 39 blood samples were taken before the start of the GFD, and 109 were taken after. Twenty control sera from healthy donors were used for comparison. Results: We found that patients had a subclinical but statistically significant increase in blood calcium, transaminases, and white blood cells, and a decrease in serum iron, at the time of diagnosis. Our study also shows that analytical values normalise within five years on a gluten-free diet. Conclusions: The use of a combination of subclinical changes, including low iron, high calcium, elevated leukocytes, lymphocytes, and ALT levels in blood samples, together with a low growth percentile, is pertinent in detecting coeliac disease. This set of parameters could help in the diagnosis of patients without clinical symptoms. We can also show that the levels of Fe, Ca, transaminases, and leucocytes remain subclinically altered after 3 years, despite the gluten-free diet

Increased Goodpasture Antigen-Binding Protein Expression Induces Type IV Collagen Disorganization and Deposit of Immunoglobulin A in Glomerular Basement Membrane

Increased expression of Goodpasture antigen-binding protein (GPBP), a protein that binds and phosphorylates basement membrane collagen, has been associated with immune complex-mediated pathogenesis. However, recent reports have questioned this biological function and proposed that GPBP serves as a cytosolic ceramide transporter (CERTL). Thus, the role of GPBP in vivo remains unknown. New Zealand White (NZW) mice are considered healthy animals although they convey a genetic predisposition for immune complex-mediated glomerulonephritis. Here we show that NZW mice developed age-dependent lupus-prone autoimmune response and immune complex-mediated glomerulonephritis characterized by elevated GPBP, glomerular basement membrane (GBM) collagen disorganization and expansion, and deposits of IgA on disrupted GBM. Transgenic overexpression of human GPBP (hGPBP) in non-lupus-prone mice triggered similar glomerular abnormalities including deposits of IgA on a capillary GBM that underwent dissociation, in the absence of an evident autoimmune response. We provide in vivo evidence that GPBP regulates GBM collagen organization and its elevated expression causes dissociation and subsequent accumulation of IgA on the GBM. Finally, we describe a previously unrecognized pathogenic mechanism that may be relevant in human primary immune complex-mediated glomerulonephritis