Influence of the Stability of a Fused Protein and Its Distance to the Amyloidogenic Segment on Fibril Formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔGD = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔGD = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation

NLRP7 in the spectrum of reproductive wastage: Rare non-synonymous variants confer genetic susceptibility to recurrent reproductive wastage

Background: NLRP7 mutations are responsible for recurrent molar pregnancies and associated reproductive wastage. To investigate the role of NLRP7 in sporadic moles and other forms of reproductive wastage, the authors sequenced this gene in a cohort of 135 patients with at least one hydatidiform mole or three spontaneous abortions; 115 of these were new patients. Methods/Results: All mutations were reviewed and their number, nature and locations correlated with the reproductive outcomes of the patients and histopathology of their products of conception. The presence of NLRP7 mutations was demonstrated in two patients with recurrent spontaneous abortions, and some rare non-synonymous variants (NSVs), present in the general population, were found to be associated with recurrent reproductive wastage. These rare NSVs were shown to be associated with lower secretion of interleukin 1β and tumour necrosis factor and therefore to have functional consequences similar to those seen in cells from patients with NLRP7 mutations. The authors also attempted to elucidate the cause of stillbirths observed in 13% of the patients with NLRP7 mutations by examining available placentas of the stillborn babies and live births from patients with mutations or rare NSVs. A number of severe to mild placental abnormalities were found, all of which are known risk factors for perinatal morbidity. Conclusions: The authors recommend close follow-up of patients with NLRP7 mutations and rare NSVs to prevent the death of the rare or reduced number of babies that reach term.link_to_subscribed_fulltex

The genomic architecture of NLRP7 is Alu rich and predisposes to disease-associated large deletions

NLRP7 is a major gene responsible for recurrent hydatidiform moles. Here, we report 11 novel NLRP7 protein truncating variants, of which five deletions of more than 1-kb. We analyzed the transcriptional consequences of four variants. We demonstrate that one large homozygous deletion removes NLRP7 transcription start site and results in the complete absence of its transcripts in a patient in good health besides her reproductive problem. This observation strengthens existing data on the requirement of NLRP7 only for female reproduction. We show that two other variants affecting the splice acceptor of exon 6 lead to its in-frame skipping while another variant affecting the splice donor site of exon 9 leads to an in-frame insertion of 54 amino acids. Our characterization of the deletion breakpoints demonstrated that most of the breakpoints occurred within Alu repeats and the deletions were most likely mediated by microhomology events. Our data define a hotspot of Alu instability and deletions in intron 5 with six different breakpoints and rearrangements. Analysis of NLRP7 genomic sequences for repetitive elements demonstrated that Alu repeats represent 48% of its intronic sequences and these repeats seem to have been inserted into the common NLRP2/7 primate ancestor before its duplication into two genes