Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r = 0.68, P = 9.2 × 10). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations inDKC1,NOP10, orNHP2cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males withDKC1p.Glu206Lys and two children with homozygousNOP10p.Thr16Met. Females with heterozygousDKC1p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin–NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin–NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafishdkc1mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

The new coronavirus disease 2019 (COVID-19) has been emerged as a rapidly spreading pandemic. The disease is thought to spread mainly from person-to-person through respiratory droplets produced when an infected person coughs, sneezes, or talks. The pathogen of COVID-19 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It infects the cells binding to the angiotensin-converting enzyme 2 receptor (ACE2) which is expressed by cells throughout the airways as targets for cellular entry. Although the majority of persons infected with SARS-CoV-2 experience symptoms of mild upper respiratory tract infection, in some people infections of the acinar airways result in severe, potentially fatal pneumonia. However, the induction of COVID-19 pneumonia requires that SARS-CoV-2 reaches the acinar airways. While huge efforts have been made to understand the spread of the disease as well as the pathogenesis following cellular entry, much less attention is paid to how SARS-CoV-2 from the environment reach the receptors of the target cells. The aim of the present study is to characterize the deposition distribution of SARS-CoV-2 in the airways upon exposure to cough-generated droplets and aerosol particles. For this purpose, the Stochastic Lung Deposition Model has been applied. Particle size distribution, breathing parameters supposing normal breathing through the nose, and viral loads were taken from the literature. We found that the probability of direct infection of the acinar airways due to inhalation of particles emitted by a bystander cough is very low. As the number of viruses deposited in the extrathoracic airways is about 7 times higher than in the acinar airways, we concluded that in most cases COVID-19 pneumonia must be preceded by SARS-CoV-2 infection of the upper airways. Our results suggest that without the enhancement of viral load in the upper airways, COVID-19 would be much less dangerous. The period between the onset of initial symptoms and the potential clinical deterioration could provide an opportunity for prevention of pneumonia by blocking or significantly reducing the transport of viruses towards the acinar airways. Therefore, even non-specific treatment forms like disinfection of the throat and nasal and oral mucosa may effectively keep the viral load of the upper airways low enough to avoid or prolong the progression of the disease. In addition, using a tissue or cloth in order to absorb droplets and aerosol particles emitted by own coughs of infected patients before re-inhalation is highly recommended even if they are alone in quarantine.

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R-and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl ؊ , 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (␣FeO 2 ) 2 -(␤Zn) 2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70 -200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl ؊ . We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.Hemoglobin (1) is a tetrameric protein, which plays a vital role in the transport of oxygen. It consists of two dimers of ␣ and ␤ subunits that reversibly bind and release oxygen (1). The description of this cooperative phenomenon has been most frequently derived from the Monod-Wyman-Changeux (MWC) 2 two-state allosteric model (2) that attributes cooperativity to a rapid equilibrium between two conformations of distinct oxygen affinity of the whole tetramer. These distinct states are the fully unliganded T-state and the fully ligated R-state. Szabo and Karplus (3) modified the two-state model incorporating the stereochemical mechanism suggested by Perutz (4) for the T to R switch, and introduced ligation-induced tertiary changes within the T-state. In this extended model (MWC-SK), it was proposed that cooperativity still works through a ligation-induced shift in the equilibrium of states T and R, but the model attributed importance in the conformational switch to certain changes at the inter-and intrasubunit interfaces.