Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

Germline gain-of-function mutations in SOS1 cause Noonan syndrome

Noonan syndrome, the most common single-gene cause of congenital heart disease, is characterized by short stature, characteristic facies, learning problems and leukemia predisposition. Gain-of-function mutations in PTPN11, encoding the tyrosine phosphatase SHP2, cause approximately 50% of Noonan syndrome cases. SHP2 is required for RAS-ERK MAP kinase (MAPK) cascade activation, and Noonan syndrome mutants enhance ERK activation ex vivo and in mice. KRAS mutations account for <5% of cases of Noonan syndrome, but the gene(s) responsible for the remainder are unknown. We identified missense mutations in SOS1, which encodes an essential RAS guanine nucleotide-exchange factor (RAS-GEF), in approximately 20% of cases of Noonan syndrome without PTPN11 mutation. The prevalence of specific cardiac defects differs in SOS1 mutation-associated Noonan syndrome. Noonan syndrome-associated SOS1 mutations are hypermorphs encoding products that enhance RAS and ERK activation. Our results identify SOS1 mutants as a major cause of Noonan syndrome, representing the first example of activating GEF mutations associated with human disease and providing new insights into RAS-GEF regulation.     

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.     

Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome

Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-L?ken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.     

Mutations in LMF1 cause combined lipase deficiency and severe hypertriglyceridemia

Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.     

The neuronal sortilin-related receptor SORL1 is genetically associated with Alzheimer disease

The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid beta peptide (Abeta) in Alzheimer disease. We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset Alzheimer disease. These variants, which occur in at least two different clusters of intronic sequences within the SORL1 gene (also known as LR11 or SORLA) may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways and that when SORL1 is underexpressed, APP is sorted into Abeta-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing Alzheimer disease.