A mutant of phosphomannomutase1 retains full enzymatic activity, but is not activated by IMP : possible implications for the disease PMM2-CDG

The most frequent disorder of glycosylation, PMM2-CDG, is caused by a deficiency of phosphomannomutase activity. In humans two paralogous enzymes exist, both of them require mannose 1,6-bis-phosphate or glucose 1,6-bis-phosphate as activators, but only phospho-mannomutase1 hydrolyzes bis-phosphate hexoses. Mutations in the gene encoding phosphomannomutase2 are responsible for PMM2-CDG. Although not directly causative of the disease, the role of the paralogous enzyme in the disease should be clarified. Phosphomannomutase1 could have a beneficial effect, contributing to mannose 6-phosphate isomerization, or a detrimental effect, hydrolyzing the bis-phosphate hexose activator. A pivotal role in regulating mannose-1phosphate production and ultimately protein glycosylation might be played by inosine monophosphate that enhances the phosphatase activity of phosphomannomutase1. In this paper we analyzed human phosphomannomutases by conventional enzymatic assays as well as by novel techniques such as 31P-NMR and thermal shift assay. We characterized a triple mutant of phospomannomutase1 that retains mutase and phosphatase activity, but is unable to bind inosine monophosphate

Proteostasis regulators modulate proteasomal activity and gene expression to attenuate multiple phenotypes in Fabry disease

The lysosomal storage disorder Fabry disease is characterized by a deficiency of the lysosomal enzyme \u3b1-Galactosidase A. The observation that missense variants in the encoding GLA gene often lead to structural destabilization, endoplasmic reticulum retention and proteasomal degradation of the misfolded, but otherwise catalytically functional enzyme has resulted in the exploration of alternative therapeutic approaches. In this context, we have investigated proteostasis regulators (PRs) for their potential to increase cellular enzyme activity, and to reduce the disease-specific accumulation of the biomarker globotriaosylsphingosine in patient-derived cell culture. The PRs also acted synergistically with the clinically approved 1-deoxygalactonojirimycine, demonstrating the potential of combination treatment in a therapeutic application. Extensive characterization of the effective PRs revealed inhibition of the proteasome and elevation of GLA gene expression as paramount effects. Further analysis of transcriptional patterns of the PRs exposed a variety of genes involved in proteostasis as potential modulators. We propose that addressing proteostasis is an effective approach to discover new therapeutic targets for diseases involving folding and trafficking-deficient protein mutants

Transcriptional regulation of the urokinase receptor (u-PAR) - A central molecule of invasion and metastasis

The phenomenon of tumor-associated proteolysis has been acknowledged as a decisive step in the progression of cancer. This short review focuses on the urokinase receptor (u-PAR), a central molecule involved in tumor-associated invasion and metastasis, and summarizes the transcriptional regulation of u-PAR. The urokinase receptor (u-PAR) is a heavily glycosylated cell surface protein and binds the serine protease urokinase specifically and with high affinity. It consists of three similar cysteine-rich repeats and is anchored to the cell membrane via a GPI-anchor. The u-PAR gene comprises 7 exons and is located on chromosome 19q13. Transcriptional activation of the u-PAR promoter region can be induced by binding of transcription factors (Sp1, AP-1, AP-2, NF-kappaB). One current study gives an example for transcriptional downregulation of u-PAR through a PEA3/ets transcriptional silencing element. Knowledge of the molecular regulation of this molecule in tumor cells could be very important for diagnosis and therapy in the near future

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with \u3b1v\u3b23 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR

Identification of trombospondin-1 as a novel amelogenin interactor by functional proteomics

Amelogenins are a set of low molecular-weight enamel proteins belonging to a group of extracellular matrix (ECM) proteins with a key role in tooth enamel development and in other regeneration processes, such as wound healing and angiogenesis. Since only few data are actually available to unravel amelogenin mechanism of action in chronic skin healing restoration, we moved to the full characterization of the human amelogenin isoform 2 interactome in the secretome and lysate of Human Umbilical Vein Endothelial cells (HUVEC), using a functional proteomic approach. Trombospondin-1 has been identified as a novel and interesting partner of human amelogenin isoform 2 and their direct binding has been validated thought biophysical orthogonal approaches

Inherited and sporadic epimutations at the IGF2-H19 locus in Beckwith-Wiedemann syndrome and Wilms' tumor

The parent-of-origin-dependent expression of IGF2 and H19 is controlled by the imprinting center 1 (IC1) consisting of a methylation-sensitive chromatin insulator. IC1 is normally methylated on the paternal chromosome and nonmethylated on the maternal chromosome. We found that 22 cases in a large cohort of patients affected by Beckwith-Wiedemann syndrome (BWS) had IC1 methylated on both parental chromosomes, resulting in biallelic activation of IGF2 and biallelic silencing of H19. These individuals had marked macrosomia and high incidence of Wilms' tumor. A subset of these patients had 1.4- to 1.8-kb deletions with hypermethylation of the remaining IC1 region and fully penetrant BWS phenotype when transmitted maternally. Another subset of individuals with IC1 hypermethylation had a similar clinical phenotype but no mutation in the local vicinity. All these cases were sporadic and in at least two families affected and unaffected members shared the same maternal IC1 allele but not the abnormal maternal epigenotype. Similarly, no IC1 deletion was detected in 10 nonsyndromic Wilms' tumors with IC1 hypermethylation. In conclusion, methylation defects at the IGF2-H19 locus can result from inherited mutations of the imprinting center and have high recurrence risk or arise independently from the sequence context and not transmitted to the progen