Characterization of novel mutations in the TEL-patch domain of the telomeric factor TPP1 associated with telomere biology disorders.

Telomeres are nucleoprotein structures that protect the chromosome ends from degradation and fusion. Telomerase is a ribonucleoprotein complex essential to maintain the length of telomeres. Germline defects that lead to short and/or dysfunctional telomeres cause telomere biology disorders (TBDs), a group of rare and heterogeneous Mendelian diseases including pulmonary fibrosis, dyskeratosis congenita, and Høyeraal-Hreidarsson syndrome. TPP1, a telomeric factor encoded by the gene ACD, recruits telomerase at telomere and stimulates its activity via its TEL-patch domain that directly interacts with TERT, the catalytic subunit of telomerase. TBDs due to TPP1 deficiency have been reported only in 11 individuals. We here report four unrelated individuals with a wide spectrum of TBD manifestations carrying either heterozygous or homozygous ACD variants consisting in the recurrent and previously described in-frame deletion of K170 (K170∆) and three novel missense mutations G179D, L184R, and E215V. Structural and functional analyses demonstrated that the four variants affect the TEL-patch domain of TPP1 and impair telomerase activity. In addition, we identified in the ACD gene several motifs associated with small deletion hotspots that could explain the recurrence of the K170∆ mutation. Finally, we detected in a subset of blood cells from one patient, a somatic TERT promoter-activating mutation that likely provides a selective advantage over non-modified cells, a phenomenon known as indirect somatic genetic rescue. Together, our results broaden the genetic and clinical spectrum of TPP1 deficiency and specify new residues in the TEL-patch domain that are crucial for length maintenance and stability of human telomeres in vivo.

AlphaFold2-guided description of CoBaHMA, a novel family of bacterial domains within the heavy-metal-associated superfamily.

Three-dimensional (3D) structure information, now available at the proteome scale, may facilitate the detection of remote evolutionary relationships in protein superfamilies. Here, we illustrate this with the identification of a novel family of protein domains related to the ferredoxin-like superfold, by combining (i) transitive sequence similarity searches, (ii) clustering approaches, and (iii) the use of AlphaFold2 3D structure models. Domains of this family were initially identified in relation with the intracellular biomineralization of calcium carbonates by Cyanobacteria. They are part of the large heavy-metal-associated (HMA) superfamily, departing from the latter by specific sequence and structural features. In particular, most of them share conserved basic amino acids  (hence their name CoBaHMA for Conserved Basic residues HMA), forming a positively charged surface, which is likely to interact with anionic partners. CoBaHMA domains are found in diverse modular organizations in bacteria, existing in the form of monodomain proteins or as part of larger proteins, some of which are membrane proteins involved in transport or lipid metabolism. This suggests that the CoBaHMA domains may exert a regulatory function, involving interactions with anionic lipids. This hypothesis might have a particular resonance in the context of the compartmentalization observed for cyanobacterial intracellular calcium carbonates.

Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection.

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis.

Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis.

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases.

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

A sequence-based foldability score combined with AlphaFold2 predictions to disentangle the protein order/disorder continuum.

Order and disorder govern protein functions, but there is a great diversity in disorder, from regions that are-and stay-fully disordered to conditional order. This diversity is still difficult to decipher even though it is encoded in the amino acid sequences. Here, we developed an analytic Python package, named pyHCA, to estimate the foldability of a protein segment from the only information of its amino acid sequence and based on a measure of its density in regular secondary structures associated with hydrophobic clusters, as defined by the hydrophobic cluster analysis (HCA) approach. The tool was designed by optimizing the separation between foldable segments from databases of disorder (DisProt) and order (SCOPe [soluble domains] and OPM [transmembrane domains]). It allows to specify the ratio between order, embodied by regular secondary structures (either participating in the hydrophobic core of well-folded 3D structures or conditionally formed in intrinsically disordered regions) and disorder. We illustrated the relevance of pyHCA with several examples and applied it to the sequences of the proteomes of 21 species ranging from prokaryotes and archaea to unicellular and multicellular eukaryotes, for which structure models are provided in the AlphaFold protein structure database. Cases of low-confidence scores related to disorder were distinguished from those of sequences that we identified as foldable but are still excluded from accurate modeling by AlphaFold2 due to a lack of sequence homologs or to compositional biases. Overall, our approach is complementary to AlphaFold2, providing guides to map structural innovations through evolutionary processes, at proteome and gene scales.

Structure basis of CFTR folding, function and pharmacology.

The root cause of cystic fibrosis (CF), the most common life-shortening genetic disease in the Caucasian population, is the loss of function of the CFTR protein, which serves as a phosphorylation-activated, ATP-gated anion channel in numerous epithelia-lining tissues. In the past decade, high-throughput drug screening has made a significant stride in developing highly effective CFTR modulators for the treatment of CF. Meanwhile, structural-biology studies have succeeded in solving the high-resolution three-dimensional (3D) structure of CFTR in different conformations. Here, we provide a brief overview of some striking features of CFTR folding, function and pharmacology, in light of its specific structural features within the ABC-transporter superfamily. A particular focus is given to CFTR's first nucleotide-binding domain (NBD1), because folding of NBD1 constitutes a bottleneck in the CFTR protein biogenesis pathway, and ATP binding to this domain plays a unique role in the functional stability of CFTR. Unraveling the molecular basis of CFTR folding, function, and pharmacology would inspire the development of next-generation mutation-specific CFTR modulators.

Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters.

Digging into the 3D Structure Predictions of AlphaFold2 with Low Confidence: Disorder and Beyond.

AlphaFold2 (AF2) has created a breakthrough in biology by providing three-dimensional structure models for whole-proteome sequences, with unprecedented levels of accuracy. In addition, the AF2 pLDDT score, related to the model confidence, has been shown to provide a good measure of residue-wise disorder. Here, we combined AF2 predictions with pyHCA, a tool we previously developed to identify foldable segments and estimate their order/disorder ratio, from a single protein sequence. We focused our analysis on the AF2 predictions available for 21 reference proteomes (AFDB v1), in particular on their long foldable segments (>30 amino acids) that exhibit characteristics of soluble domains, as estimated by pyHCA. Among these segments, we provided a global analysis of those with very low pLDDT values along their entire length and compared their characteristics to those of segments with very high pLDDT values. We highlighted cases containing conditional order, as well as cases that could form well-folded structures but escape the AF2 prediction due to a shallow multiple sequence alignment and/or undocumented structure or fold. AF2 and pyHCA can therefore be advantageously combined to unravel cryptic structural features in whole proteomes and to refine predictions for different flavors of disorder.

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators.

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin-Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

Molecular mechanisms of cystic fibrosis - how mutations lead to misfunction and guide therapy.

Cystic fibrosis, the most common autosomal recessive disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride and bicarbonate channel that regulates ion and water transport in secretory epithelia. Although all mutations lead to the lack or reduction in channel function, the mechanisms through which this occurs are diverse - ranging from lack of full-length mRNA, reduced mRNA levels, impaired folding and trafficking, targeting to degradation, decreased gating or conductance, and reduced protein levels to decreased half-life at the plasma membrane. Here, we review the different molecular mechanisms that cause cystic fibrosis and detail how these differences identify theratypes that can inform the use of directed therapies aiming at correcting the basic defect. In summary, we travel through CFTR life cycle from the gene to function, identifying what can go wrong and what can be targeted in terms of the different types of therapeutic approaches.

Severity of the S1251N allele in cystic fibrosis is affected by the presence of the F508C variant in cis.

BACKGROUND: In cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation. METHODS: Genetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated. RESULTS: Organoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation. CONCLUSIONS: In vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF.

Gain-of-function IKZF1 variants in humans cause immune dysregulation associated with abnormal T/B cell late differentiation.

IKZF1/IKAROS is a key transcription factor of lymphocyte development expressed throughout hematopoiesis. Heterozygous germline IKZF1 haploinsufficient (IKZF1HI) and dominant-negative (IKZF1DN) variants in humans cause B cell immune deficiency and combined immunodeficiency. Here, we identified previously unidentified heterozygous IKZF1 variants (R183C/H) located in the DNA binding domain in eight individuals with inflammatory, autoimmune, allergic symptoms, and abnormal plasma cell (PC) proliferation. Leukocytes of patients exhibited specific defects including impaired IL-2 production by T cells, T helper (TH) skewing toward TH2, low numbers of regulatory T cells (Treg), eosinophilia, and abnormal PC proliferation. In contrast to IKZF1HI and IKZF1DN, IKZF1R183H/C proteins showed increased DNA binding associated with increased gene expression of TH2 and PC differentiation, thus demonstrating that IKZF1R183H/C behave as gain-of-function (GOF) alleles. In vitro treatment with lenalidomide, known to degrade IKZF1, corrected TH2 and PC abnormalities caused by IKZF1R183H/C. These data extend the spectrum of pathological mechanisms associated with IKZF1 deficiencies and highlight the role of IKZF1 in late lymphoid differentiation stages.

A New Gene Family Diagnostic for Intracellular Biomineralization of Amorphous Ca Carbonates by Cyanobacteria.

Cyanobacteria have massively contributed to carbonate deposition over the geological history. They are traditionally thought to biomineralize CaCO3 extracellularly as an indirect byproduct of photosynthesis. However, the recent discovery of freshwater cyanobacteria-forming intracellular amorphous calcium carbonates (iACC) challenges this view. Despite the geochemical interest of such a biomineralization process, its molecular mechanisms and evolutionary history remain elusive. Here, using comparative genomics, we identify a new gene (ccyA) and protein family (calcyanin) possibly associated with cyanobacterial iACC biomineralization. Proteins of the calcyanin family are composed of a conserved C-terminal domain, which likely adopts an original fold, and a variable N-terminal domain whose structure allows differentiating four major types among the 35 known calcyanin homologs. Calcyanin lacks detectable full-length homologs with known function. The overexpression of ccyA in iACC-lacking cyanobacteria resulted in an increased intracellular Ca content. Moreover, ccyA presence was correlated and/or colocalized with genes involved in Ca or HCO3- transport and homeostasis, supporting the hypothesis of a functional role of calcyanin in iACC biomineralization. Whatever its function, ccyA appears as diagnostic of intracellular calcification in cyanobacteria. By searching for ccyA in publicly available genomes, we identified 13 additional cyanobacterial strains forming iACC, as confirmed by microscopy. This extends our knowledge about the phylogenetic and environmental distribution of cyanobacterial iACC biomineralization, especially with the detection of multicellular genera as well as a marine species. Moreover, ccyA was probably present in ancient cyanobacteria, with independent losses in various lineages that resulted in a broad but patchy distribution across modern cyanobacteria.

Inherited human Apollo deficiency causes severe bone marrow failure and developmental defects.

Inherited bone marrow failure syndromes (IBMFSs) are a group of disorders typified by impaired production of 1 or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant, Høyeraal-Hreidarsson (HH) syndrome, are rare IBMFSs characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. We identified biallelic variants in the gene encoding the 5'-to-3' DNA exonuclease Apollo/SNM1B in 3 unrelated patients presenting with a DC/HH phenotype consisting of early-onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly, and/or intrauterine growth retardation. All 3 patients carry a homozygous or compound heterozygous (in combination with a null allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080 cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS that combines clinical hallmarks of DC/HH with normal telomere length.

Intergenic ORFs as elementary structural modules of de novo gene birth and protein evolution.

The noncoding genome plays an important role in de novo gene birth and in the emergence of genetic novelty. Nevertheless, how noncoding sequences' properties could promote the birth of novel genes and shape the evolution and the structural diversity of proteins remains unclear. Therefore, by combining different bioinformatic approaches, we characterized the fold potential diversity of the amino acid sequences encoded by all intergenic open reading frames (ORFs) of S. cerevisiae with the aim of (1) exploring whether the structural states' diversity of proteomes is already present in noncoding sequences, and (2) estimating the potential of the noncoding genome to produce novel protein bricks that could either give rise to novel genes or be integrated into pre-existing proteins, thus participating in protein structure diversity and evolution. We showed that amino acid sequences encoded by most yeast intergenic ORFs contain the elementary building blocks of protein structures. Moreover, they encompass the large structural state diversity of canonical proteins, with the majority predicted as foldable. Then, we investigated the early stages of de novo gene birth by reconstructing the ancestral sequences of 70 yeast de novo genes and characterized the sequence and structural properties of intergenic ORFs with a strong translation signal. This enabled us to highlight sequence and structural factors determining de novo gene emergence. Finally, we showed a strong correlation between the fold potential of de novo proteins and one of their ancestral amino acid sequences, reflecting the relationship between the noncoding genome and the protein structure universe.

In vitro functional rescue by ivacaftor of an ABCB11 variant involved in PFIC2 and intrahepatic cholestasis of pregnancy.

BACKGROUND: ABCB11 variations are responsible for a spectrum of rare liver diseases, including progressive familial intrahepatic cholestasis type 2 (PFIC2) and intrahepatic cholestasis of pregnancy (ICP). Current medical treatment of these conditions mostly relies on ursodeoxycholic acid with limited efficacy. We report on the in vitro study of the p.A257V missense variant of ABCB11 identified in a PFIC2 patient and in her mother who experienced ICP. RESULTS: The Ala257 residue is located outside the ATP-binding site of ABCB11. We show that the p.A257V variant of ABCB11 is correctly expressed at the canalicular membrane of HepG2 cells but that its function significantly decreased when studied in MDCK cells. This functional defect can be fully rescued by Ivacaftor. CONCLUSION: Ivacaftor could be considered as a new pharmacological tool able to respond to an unmet medical need for patients with ICP and PFIC2 due to ABCB11 variations affecting ABCB11 function, even when the residue involved is not located in an ATP-binding site of ABCB11.

Structure-Based Understanding of ABCA3 Variants.

ABCA3 is a crucial protein of pulmonary surfactant biosynthesis, associated with recessive pulmonary disorders such as neonatal respiratory distress and interstitial lung disease. Mutations are mostly private, and accurate interpretation of variants is mandatory for genetic counseling and patient care. We used 3D structure information to complete the set of available bioinformatics tools dedicated to medical decision. Using the experimental structure of human ABCA4, we modeled at atomic resolution the human ABCA3 3D structure including transmembrane domains (TMDs), nucleotide-binding domains (NBDs), and regulatory domains (RDs) in an ATP-bound conformation. We focused and mapped known pathogenic missense variants on this model. We pinpointed amino-acids within the NBDs, the RDs and within the interfaces between the NBDs and TMDs intracellular helices (IHs), which are predicted to play key roles in the structure and/or the function of the ABCA3 transporter. This theoretical study also highlighted the possible impact of ABCA3 variants in the cytosolic part of the protein, such as the well-known p.Glu292Val and p.Arg288Lys variants.