Oligomerization Sites Research Articles

Molecular and thermodynamic determinants of self-assembly and hetero-oligomerization in the enterobacterial thermo-osmo-regulatory protein H-NS.

Environmentally regulated gene expression is critical for bacterial survival under stress conditions, including extremes in temperature, osmolarity and nutrient availability. Here, we dissect the thermo- and osmo-responsory behavior of the transcriptional repressor H-NS, an archetypal nucleoid-condensing sensory protein, ubiquitous in enterobacteria that infect the mammalian gut. Through experiments and thermodynamic modeling, we show that H-NS exhibits osmolarity, temperature and concentration dependent self-association, with a highly polydisperse native ensemble dominated by monomers, dimers, tetramers and octamers. The relative population of these oligomeric states is determined by an interplay between dimerization and higher-order oligomerization, which in turn drives a competition between weak homo- versus hetero-oligomerization of protein-protein and protein-DNA complexes. A phosphomimetic mutation, Y61E, fully eliminates higher-order self-assembly and preserves only dimerization while weakening DNA binding, highlighting that oligomerization is a prerequisite for strong DNA binding. We further demonstrate the presence of long-distance thermodynamic connectivity between dimerization and oligomerization sites on H-NS which influences the binding of the co-repressor Cnu, and switches the DNA binding mode of the hetero-oligomeric H-NS:Cnu complex. Our work thus uncovers important organizational principles in H-NS including a multi-layered thermodynamic control, and provides a molecular framework broadly applicable to other thermo-osmo sensory proteins that employ similar mechanisms to regulate gene expression.

Structural investigation of Amphibalanus amphitrite cement proteins: an in silico study

Balanomorpha, commonly known as barnacles, are leading biofouling animals belonging to subclass Cirripedia that adhere durably to different submerged surfaces by utilizing a chiefly proteinaceous cement. According to prior experiments, adhesion is most likely made possible by the self-assembling aggregates reputed as amyloid-like nanofibers. The secreted cement contains numerous proteins, among which CP19k and CP20k are thought to have a substantial influence on the adhesion process. The molecular configuration and atomistic interactions that result in this firm cement are not yet completely understood. Herein, artificial-intelligence-based structure prediction and molecular dockings were used to inspect the potential role of the AaCP19k and AaCP20k-1 of Amphibalanus amphitrite in the formation of amyloid-like nanofibers. The anticipated structure of AaCP19k was highly accurate, and its β-sandwich folding had a close resemblance to cross-β motifs found in amyloid nanofibers. In AaCP19k, β1–2 and β7–8 act as oligomerization sites where stable dimers and trimers can be assembled. These modeled oligomerization interfaces point to the self-assembly site through which fibrillization might happen. The structural flexibility of AaCP20k-1 yielded low-accuracy models, but a conserved β-hairpin and an α-helix were evident with high confidence. These structural properties can be employed in prospective studies to develop bioadhesives and design antifouling substances.

Structural Basis of Clostridium perfringens Enterotoxin Activation and Oligomerization by Trypsin.

Clostridium perfringens enterotoxin (CpE) is a β-pore forming toxin that disrupts gastrointestinal homeostasis in mammals by binding membrane protein receptors called claudins. Although structures of CpE fragments bound to claudins have been determined, the mechanisms that trigger CpE activation and oligomerization that lead to the formation of cytotoxic β-pores remain undetermined. Proteolysis of CpE in the gut by trypsin has been shown to play a role in this and subsequent cytotoxicity processes. Here, we report solution structures of full-length and trypsinized CpE using small-angle X-ray scattering (SAXS) and crystal structures of trypsinized CpE and its C-terminal claudin-binding domain (cCpE) using X-ray crystallography. Mass spectrometry and SAXS uncover that removal of the CpE N-terminus by trypsin alters the CpE structure to expose areas that are normally unexposed. Crystal structures of trypsinized CpE and cCpE reveal unique dimer interfaces that could serve as oligomerization sites. Moreover, comparisons of these structures to existing ones predict the functional implications of oligomerization in the contexts of cell receptor binding and β-pore formation. This study sheds light on trypsin's role in altering CpE structure to activate its function via inducing oligomerization on its path toward cytotoxic β-pore formation. Its findings can incite new approaches to inhibit CpE-based cytotoxicity with oligomer-disrupting therapeutics.

A conserved oligomerization domain in the disordered linker of coronavirus nucleocapsid proteins.

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

TIFA promotes colorectal cancer cell proliferation in an RSK- and PRAS40-dependent manner.

Previous studies have reported that TIFA plays different roles in various tumor types. However, the function of TIFA in colorectal cancer (CRC) remains unclear. Here, we showed that the expression of TIFA was markedly increased in CRC versus normal tissue, and positively correlated with CRC TNM stages. In agreement, we found that the CRC cell lines show increased TIFA expression levels versus normal control. The knockdown of TIFA inhibited cell proliferation but had no effect on cell apoptosis in vitro or in vivo. Moreover, the ectopic expression of TIFA enhanced cell proliferation ability in vitro and in vivo. In contrast, the expression of mutant TIFA (T9A, oligomerization site mutation; D6, TRAF6 binding site deletion) abolished TIFA‐mediated cell proliferation enhancement. Exploration of the underlying mechanism revealed that the protein synthesis‐associated kinase RSK and PRAS40 activation were responsible for TIFA‐mediated CRC progression. In summary, these findings suggest that TIFA plays a role in mediating CRC progression. This could provide a promising target for CRC therapy.

MO044: Cellular mechanism of the exceptional dominant transmission in NPHS2-associated glomerulopathy

Abstract BACKGROUND AND AIMS Mutations of NPHS2 cause autosomal recessive steroid-resistant nephrotic syndrome (SRNS). The encoded podocin, a key component of the slit diaphragm, homo-oligomerizes through two C-terminal oligomerization sites. We formerly found that specific podocin variants may exert a dominant negative effect against R229Q podocin variant secondary to an altered oligomerization [1, 2]. Recently, two families with autosomal dominant focal segmental glomerulosclerosis were found to carry a de novo heterozygous NPHS2 last-exon frameshift mutations affecting the second oligomerization site (either c.989_992delTGTC, p.L330Pfs*17 or c.988_989delCT, p.L330Vfs*15) [3]. We hypothesized that these podocin variants exert a dominant negative effect against wild type (WT) podocin. METHODS We coexpressed eGFP-tagged WT podocin with either hemagglutinin-tagged (HA) WT, p.L330Pfs*17 or p.L330Vfs*15 podocin variants in cultured podocytes. Plasma membrane was stained with CF405M-conjugated wheat germ agglutinin (WGA), and immunohistochemical staining was performed with primary anti-HA and secondary AlexaFluor568-conjugated antibodies. All analysis was done in a blinded fashion. The membrane-targeting of podocin was assessed by the ratio of the WGA-labeled perimembranous area that colocalized with podocin (Mander's overlap coefficients, MOC) and the colocalization of the coexpressed variants were estimated as Pearson correlation coefficients (PCC) by FiJi software on confocal microscopic images. MOC and PCC were compared by Holmes–Bonferroni adjusted Wilcoxon signed-rank test. RESULTS The frameshift podocin variants were not membranous in monoexpression [MOC: HA-p.L330Pfs*17 (n = 26): 0.08 (0.03–0.22) median (interquartile ranges); HA-p.L330Vfs*15 (n = 24): 0.07 (0.02–0.16)]. The GFP-tagged WT podocin strongly colocalized with the HA-tagged WT or frameshift podocin variants [PCC: WT-eGFP + HA-WT (n = 19): 0.64 (0.49–0.71); WT-eGFP + HA-L330Pfs*17 (n = 17): 0.54 (0.35–0.58); WT-eGFP + HA-L330Vfs*15 (n = 17): 0.44 (0.29–0.66)]. The GFP-WT podocin was retained in intracellular compartments in the presence of any of the two frameshift podocin variants [MOCWT(WT) = 0.58 (0.46–0.69)], MOCWT(L330Pfs*17) = 0.01 (0.002–0.029), P = 2x10-6 versus WT(WT); MOCWT(L330Vfs*15) = 0.003 (0.001–0.013), P = 2 x 10−6 versus WT(WT)]. CONCLUSIONS The dominant podocin variants retain WT podocin in intracellular compartments explaining the exceptional dominant transmission of NPHS2-associated glomerulopathy. This is in accordance with our former results showing the primary role of C-terminal hetero-oligomerization in exerting interallelic interactions of NPHS2 alleles. FUNDING MTA-SE Lendulet Research Grant (LP2015-11/2015), OTKA KH125566 and K135798, and ÚNKP-20–3-I-SE-29 from the source of the National Research, Development and Innovation Fund.

The first structure-function study of GH151 α-l-fucosidase uncovers new oligomerization pattern, active site complementation, and selective substrate specificity.

Fucosylated compounds are abundantly present in nature and are associated with many biological processes, therefore carrying great potential for use in medicine and biotechnology. Efficient ways to modify fucosylated compounds are still being developed. Promising results are provided by glycosyl hydrolases with transglycosylating activities, such as α-l-fucosidase isoenzyme 2 from Paenibacillus thiaminolyticus (family GH151 of Carbohydrate-Active enZYmes). Currently, there is no 3D structure representing this glycoside hydrolase family and only a few members have been investigated. Here, we present the first structure-function study of a GH151 member, providing the key insights into its specific oligomerization and active site properties. According to the crystal structure, small-angle X-ray scattering data and catalytic investigation, this enzyme functions as a tetramer of a new type and represents the second known case of active site complementation among all α-l-fucosidases. Mutation of the active site-complementing residue histidine 503 to alanine confirmed its influence on α-l-fucosidase activity and, specifically, on substrate binding. Several unique features of GH151 family α-l-fucosidases were revealed, including the oligomerization pattern, active site accessibility and complementation, and substrate selectivity. Some common properties of GH151 glycosyl hydrolases then would be the overall three-domain structure and conservation of the central domain loop 2 function, including its complementation role and the formation of the carbohydrate-binding platform in the active site vicinity.

Identification of the structure of Ni active sites for ethylene oligomerization on an amorphous silica-alumina supported nickel catalyst

The structure of Ni active sites supported on amorphous silica-alumina supports with different contents of Al2O3 loadings in relation to their activities in ethylene oligomerization were investigated. Two kinds of Ni sites were detected by in situ FTIR-CO and H2-TPR experiments, that are Ni2+ cations as grafted on weak acidic silanols and Ni2+ cations at ion-exchange positions. The ethylene oligomerization activities of these Ni/ASA catalysts were found an ascending tendency as the Al2O3 loading decreased, which could be attributed to the enriched concentration of Ni2+ species on acidic silanols with a weaker interaction with the amorphous silica-alumina support. These Ni2+ species were more easily to be evolved into Ni+ species, which has been identified to be the active sites of ethylene oligomerization. Thus, it seems reasonable to conclude that Ni2+ species grafted on acidic silanols were the precursors of active sites.

The N-terminal region of Jaw1 has a role to inhibit the formation of organized smooth endoplasmic reticulum as an intrinsically disordered region

Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP3R3) and SUN2. In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.

Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gram-negative bacteria. LPS is translocated to the OM by the LPS transport (Lpt) system. In the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LPS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmid-mediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. Complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. Finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability.

Insights Into the Mechanism of MCT8 Oligomerization.

Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in MCT8 deficiency, characterized by severe intellectual and motor disability. The MCT8 protein is predicted to have 12 transmembrane domains (TMDs) and is expressed as monomers, homodimers, and homo-oligomers. This study aimed to delineate the mechanism of MCT8 oligomerization. Coimmunoprecipitation studies demonstrated that lithium dodecyl sulfate effectively disrupts MCT8 protein complexes, indicating the involvement of non-covalent interactions. Successive C-terminal truncations of the MCT8 protein altered the oligomerization pattern only if introduced in the N-terminal half of the protein (TMD1-6). The truncation at extracellular loop 1 (E206X) still allowed homodimerization, but completely abrogated homo-oligomerization, whereas both were preserved by the C231X mutant (at TMD2), suggesting that the minimally required oligomerization sites are located proximal of Cys231. However, mutant constructs lacking the intracellular N-terminus or TMD1 and 2 were still capable to form homo-oligomers. Therefore, other domains distal of Cys231 are also likely to be involved in the formation of extensive multidomain interactions. This hypothesis was supported by structural modeling. Despite multiple approaches, MCT8 oligomerization could not be fully abrogated unless a substantial part of the protein was removed, precluding detailed studies into its functional role. Together, our findings suggest that MCT8 oligomerization involves extensive noncovalent interactions between the N-terminal halves of MCT8 proteins. Most mutations identified in patients with MCT8 deficiency have only minor effects on MCT8 oligomerization and, thus, impaired oligomerization does not appear to be an important pathogenic mechanism.

Biochemistry of prenylated-FMN enzymes.

The reversible (de)carboxylation of unsaturated carboxylic acids is carried out by the UbiX-UbiD system, ubiquitously present in microbes. The biochemical basis of this challenging reaction has recently been uncovered by the discovery of the UbiD cofactor, prenylated FMN (prFMN). This heavily modified flavin is synthesized by the flavin prenyltransferase UbiX, which catalyzes the non-metal dependent prenyl transfer from dimethylallyl(pyro)phosphate (DMAP(P)) to the flavin N5 and C6 positions, creating a fourth non-aromatic ring. Following prenylation, prFMN undergoes oxidative maturation to form the iminium species required for UbiD activity. prFMNiminium acts as a prostethic group and is bound via metal ion mediated interactions between UbiD and the prFMNiminium phosphate moiety. The modified isoalloxazine ring is place adjacent to the E(D)-R-E UbiD signature sequent motif. The fungal ferulic acid decarboxylase Fdc from Aspergillus niger has emerged as a UbiD-model system, and has yielded atomic level insight into the prFMNiminium mediated (de)carboxylation. A wealth of data now supports a mechanism reliant on reversible 1,3 dipolar cycloaddition between substrate and cofactor for this enzyme. This poses the intriguing question whether a similar mechanism is used by all UbiD enzymes, especially those that act as carboxylases on inherently more difficult substrates such as phenylphosphate or benzene/naphthalene. Indeed, considerable variability in terms of oligomerization, domain motion and active site structure is now reported for the UbiD family.

Quantitative Relationship Between Activity and Acid Site Distribution in the Oligomerization of Ethylene Over MCM-41 Catalyst

Nickel ion exchanged MCM-41 samples with different Si/Al ratios and Ni-ZSM-5/MCM-41 were prepared and used to catalyze ethylene oligomerization which was performed in a fixed bed model (T = 523 K, P = 2 MPa). A temperature programmed desorption of ammonia (NH3-TPD) method was employed to quantitatively determine the acid strength distribution of Ni-MCM-41 samples with different Si/Al ratios and Ni-ZSM-5/MCM-41. The TPD spectrum was analyzed by an interrupted-TPD method with desorption-control (dc-method) to obtain the desorption activation energy of ammonia, which can be used as acid strength scale for the purpose of correlation with catalytic activity. A quantitative relationship between catalyst acidity and catalytic activity was established by a Bronsted type equation. The site with desorption activation energy of ammonia E = 124 kJ mol−1 was found as the most suitable site for ethylene oligomerization. An approximate linear relationship is obtained between the activity of the ethylene oligomerization reaction and the acid amount corresponding to the acid site with E = 124 kJ mol−1.

Structural Evidence for an Octameric Ring Arrangement of SARM1

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome—well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM1–2 domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM1–2 and found that it also forms a closed octameric ring in the crystal lattice. The SAM1–2 ring interactions are mediated by complementing “lock and key” hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM1–2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development.

A kinetic study of the NO to NO2 oxidation mechanism over Fe-FER: A combined analysis of operando surface and gas phase data

Abstract Diesel engines when compared with gasoline engines produce higher amount of NOx due the operating conditions. Among the NOx produced NO represents the great majority while, under air excess conditions, the removal of NOx is necessarily catalytic and still challenging. In the ammonia based SCR, it is often admitted that, when dealing with the original formulation developed for stationary sources (V/TiO2), the fast SCR proceeds in presence of an equimolar mixture of NO and NO2. Most of the catalytic formulations developed up to now (except Cu based zeolites) thus suffer the same rate determining step consisting in the NO to NO2 oxidation. Aiming at improving the catalytic efficiency through the catalyst composition, it is fundamental to understand the reaction mechanism at its elementary steps in order to properly design the active sites. The choice regarding the distinct possible hypothetical mechanisms was thus here guided by the nature of the active sites. It is indeed worth knowing, for a better tuning of the catalyst formulation, whether isolated, oligomeric or dual iron sites are involved in the catalytic loop. The approach in this work consists in a systematic study of the influence of the inlet NO concentration under O2 excess on both the initial reaction rate (NO to NO2 conversion level kept below 10%) and the NO coverage level onto iron measured by operando FTIR. Isothermal experiments were performed over an aged FeFER catalyst at three distinct temperatures. The whole set of data was then processed and compared to the expected evolution derived from five possible mechanisms. The best mechanism determined in the frame of this study involves isolated iron sites onto which NO and O2 co-adsorb and the corresponding rate determining step consists in the dissociation of the so-formed Fe_NOO2 intermediate species. The associated activation energy and reaction enthalpy values are then evaluated.

The Effect of the Active-Site Structure on the Activity of Copper Mordenite in the Aerobic and Anaerobic Conversion of Methane into Methanol.

Samples of the zeolite mordenite with different Si/Al ratios were used to synthesize materials with monomeric and oligomeric copper sites that are active in the direct conversion of methane into methanol. A comparison of two reactivation protocols with oxygen (aerobic oxidation) and water (anaerobic oxidation), respectively, revealed that such copper-oxo species possess different reactivity towards methane and water. We show for the first time that oligomeric copper species exhibit high activity under both aerobic and anaerobic activation conditions, whereas monomeric copper sites produce methanol only in aerobic processes.

The Effect of the Active‐Site Structure on the Activity of Copper Mordenite in the Aerobic and Anaerobic Conversion of Methane into Methanol

AbstractSamples of the zeolite mordenite with different Si/Al ratios were used to synthesize materials with monomeric and oligomeric copper sites that are active in the direct conversion of methane into methanol. A comparison of two reactivation protocols with oxygen (aerobic oxidation) and water (anaerobic oxidation), respectively, revealed that such copper–oxo species possess different reactivity towards methane and water. We show for the first time that oligomeric copper species exhibit high activity under both aerobic and anaerobic activation conditions, whereas monomeric copper sites produce methanol only in aerobic processes.

H2O2-based selective epoxidations: Nb-silicates versus Ti-silicates

This paper aims at a comparison of the stability and catalytic performance of niobium- and titanium-containing mesoporous silicates prepared by evaporation-induced self-assembly (EISA) in selective oxidations with hydrogen peroxide. The catalysts comprised evenly dispersed dimeric and/or small oligomeric active sites (MNb(V) or Ti(IV)), and the Nb sites revealed better hydrolytic stability than the Ti ones, as demonstrated by DRS UV–vis. An attempt has been made to rationalize the differences observed in chemo- and regioselectivity of alkene epoxidation using kinetic modeling tools. The rate law established for cyclooctene epoxidation over the Nb- and Ti-catalysts is consistent with an Eley–Rideal mechanism that involves adsorption of H2O2 on M sites, interaction between M and H2O2 to afford a hydroperoxo species ‘MOOH’ and water, followed by oxygen transfer from ‘MOOH’ to CC bond, producing epoxide and regenerating M. The higher epoxidation (heterolytic pathway) selectivity of the Nb catalysts is, at least partially, related to their lower activity in H2O2 unproductive decomposition. The apparent activation energy of H2O2 degradation is higher for Nb-silicate (20.5 vs 14.8 kcal/mol for Ti-silicate), while the activation energies of epoxidation follow an opposite trend (11.9 vs 14.2 kcal/mol). The heterolytic pathway selectivity of Nb catalysts can be greatly improved by decreasing the reaction temperature. The difference in regioselectivity of limonene epoxidation over Nb- and Ti-silicates observed in MeCN, where the nature of the catalysis is truly heterogeneous, indicates different structures of the active ‘NbOOH’ and ‘TiOOH’ species and/or different mechanisms of the oxygen transfer step. The inversion of regioselectivity observed for Nb catalyst in MeOH may be caused by Nb leaching in this solvent and/or changing the structure of ‘NbOOH’.

C-terminal oligomerization of podocin mediates interallelic interactions

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 (r2 = 0.68, P = 9.2 × 10−32). 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.

Functionally Significant Amino Acid Motifs of Heat Shock Proteins: Structural and Bioinformatics Analyses of Hsp60/Hsp10 in Five Classes of Chordata

The Hsp60/Hsp10 chaperonin system is one of the most studied systems of cell emergency responses to stresses associated with changes in environmental conditions. In this regard, we have performed a bioinformatics analysis of 164 amino acid sequences of Hsp60 and 125 amino acid sequences of Hsp10 in five classes of chordata. This enabled uncovering the relationship between the identity of the amino acid composition of Hsp60/Hsp10 and the evolutionary distance between classes of chordata. In the course of the study of the chaperonin crystal structure, potentially significant amino acid motifs responsible for the oligomerization of Hsp60 and Hsp10 monomers and the association/dissociation of the Hsp60 and Hsp10 heterodimer have been identified. In addition, we have established that Hsp60 and Hsp10 can form amyloid fibrils due to structural features through the alternative using of the oligomerization sites of monomers as well as association/dissociation sites.