Biology subjects

Modenutti, C. P.

Publications and source records attributed to Modenutti, C. P..

10 recordsLinked to original sources

bioRxiv2025

A cryptic pocket allosterically modulates oligosaccharide binding to DC-SIGN

DC-SIGN is a C-type lectin receptor expressed on antigen-presenting cells, crucial for pathogen recognition and immune modulation. Here, we identify and characterize a previously unrecognized cryptic allosteric pocket in DC-SIGN using molecular dynamics simulations, NMR spectroscopy, cryogenic electron microscopy and biochemical assays. Rotation of the gatekeeper residue M270 exposes the pocket whose occupancy modulates glycan binding. Mutations M270F and T314A mimic the occupied and unoccupied states of this pocket, respectively, shifting the conformational equilibrium of -helix 2 and altering oligosaccharide affinity via the extended carbohydrate binding site. While Ca{superscript 2} coordination at the canonical binding site remains unaffected, our data reveal a complex interplay between the Ca{superscript 2} binding sites and the canonical and extended glycan binding surfaces. These findings uncover a hierarchical allosteric mechanism that enables selective tuning of glycan affinity and suggest the cryptic pocket as a novel target for drug discovery in C-type lectins.

biochemistry↗

bioRxiv2025

Structure and function of the EDEM:PDI ERAD checkpoint complex

The ERAD-L checkpoint complex de-mannosylates misfolded glycoproteins with lumenal defects, targeting them to retrotranslocation, ubiquitination, and proteasomal degradation. Commitment to ERAD-L requires an Endoplasmic Reticulum-Degradation Enhancing alpha-Mannosidase-like protein (EDEM) and its associated Protein Disulfide Isomerase (PDI). We determined Cryo-EM structures of the Chaetomium thermophilum EDEM:PDI heterodimer, both by itself and in complex with a classic ERAD substrate, the alpha1-antitrypsin Null Hong Kong mutant (A1AT-NHK). The EDEM catalytic domain nestles within the PDI arc. One intermolecular disulfide, between the a' domain of PDI and the first conserved cysteine of the EDEM linker (Cys A), stably links the two proteins. A second intermolecular disulfide, between the second conserved cysteine of the EDEM linker (Cys B) and the PDI a domain, stabilises the compact apo conformation. In the substrate-bound complex, the Cys B intermolecular disulfide is reduced and the catalytic CXXC motif of the PDI a domain oxidised. Release of the Cys B linkage increases the conformational freedom of the EDEM linker, allowing the EDEM C-terminal domains to adopt both proximal and distal conformations relative to the catalytic domain. The structures provide a framework for discovery of EDEM:PDI modulators, with potential applications in virology, rare genetic disease and cancer, and as reagents for glycoprotein quality-control engineering.

biochemistry↗

bioRxiv2023

Yerba mate (Ilex paraguariensis) genome provides new insights into convergent evolution of caffeine biosynthesis

Yerba mate (Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large yerba mate genome size is partly due to a whole-genome duplication (Ip-) during the early evolutionary history of Ilex, in addition to the hexaploidization event ({gamma}) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of its relatives, coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in yerba mate and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the x-ray diffraction data suggests structural constraints are minimal for the convergent evolution of individual reactions.

plant biology↗

bioRxiv2023

Hidden Markov Models based search in combination with structural bioinformatics pipeline leads to the identification of DAF-12 distant orthologous in Meloidogyne incognita

Root-knot nematode (RKN) Meloidogyne spp. is one of the most damaging parasites due to its wide range of hosts. Here, we report a C. elegans receptor DAF-12 ortholog gene in Meloidogyne incognita (DAF-12Minc), a promising molecular target to modify the RKN life cycle. Using a combination of Hidden Markov Models (HMM) based sequence search and phylogenetic analysis we identified three DAF-12Minc genes. Although the global sequence identity between previously reported DAF-12 genes and DAF-12Minc was acceptable, the correlation between binding site residues was low in the multiple sequence alignment (MSA). Since those residues are critical for DAF-12 interaction with its ligand, the dafachronic acids (DAs), and thus its biological role, we investigated whether even if the sequence conservation is low, the active site structure was conserved and thus able to bind DAs. For this purpose, we built accurate homology models of DAF-12Minc and used them to identify and characterize the ligand binding site (LBS) and its molecular interactions with DAs-like compounds. Finally, we cloned, expressed, and evaluated the biological role of DAF-12Minc in vitro and in vivo using a DAF-12 antagonist. These in vivo results suggest that our strategy was effective to find orthologous genes among species even when sequence similarity is low. Author summaryRoot-knot nematodes are parasitic to plants and responsible for causing a significant loss of millions of dollars every year in crops worldwide, which makes it necessary to develop effective strategies to combat them. One popular approach is to identify genes that can serve as molecular targets. Typically, such molecular targets are discovered through basic research on model organisms. However, since they can be quite different from the target organism, conventional tools may not always be efficient in extrapolating results. Dafachronic acids (DAs) are a crucial class of steroid hormones that regulate the development and physiology of nematodes. They are synthesized from cholesterol and are controlled by a nuclear hormone receptor known as DAF-12. This receptor acts as a master regulator of gene expression, playing a vital role in nematode biology, including development, reproduction, metabolism, stress response, and longevity. Therefore, DAF-12 is a promising molecular target for controlling parasitic nematodes. Although DAF-12 was initially discovered in the model organism Caenorhabditis elegans and subsequently found in some parasitic nematodes, previous attempts to identify molecular targets in the Meloidogyne genus failed to detect DAF-12 orthologs. To address this gap, we employed a combination of sequence and structure analysis to identify potential candidates for DAF-12, a known and validated molecular target, which had not yet been found in Meloidogyne incognita. Our bioinformatics predictions were experimentally validated, which may serve as a starting point for future campaigns aimed at developing parasite control strategies based on this relevant molecular target.

bioinformatics↗

bioRxiv2023

Molecular Dynamics and Water site bias docking method allows the identification of key amino acids in the Carbohydrate Recognition Domain of a viral protein

Carbohydrate-binding modules (CBMs) are protein domains that typically reside near catalytic domains, increasing substrate-protein proximity by constraining the conformational space of carbohydrates. Due to the flexibility and variability of glycans, the molecular details of how these protein regions recognize their target molecules are not always fully understood. Computational methods, including molecular docking and molecular dynamics simulations, have been employed to investigate lectin-carbohydrate interactions. In this study, we introduce a novel approach that integrates multiple computational techniques to identify the critical amino acids involved in the interaction between a CBM located at the tip of bacteriophage J-1s tail and its carbohydrate counterparts. Our results highlight three amino acids that play a significant role in binding, which we confirmed through in vitro experiments. By presenting this approach, we offer an intriguing alternative for pinpointing amino acids that contribute to protein-sugar interactions, leading to a more thorough comprehension of the molecular determinants of lectin-carbohydrate interactions.

bioinformatics↗

bioRxiv2023

Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells

Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity ("responsive mutant"). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1-/-and UGGT1/2-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. SynopsisDeletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1-/- single and UGGT1/2-/- double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate. O_FIG O_LINKSMALLFIG WIDTH=120 HEIGHT=200 SRC="FIGDIR/small/542711v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@ac29f1org.highwire.dtl.DTLVardef@f5bcbforg.highwire.dtl.DTLVardef@13ab5a1org.highwire.dtl.DTLVardef@16a9406_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 WY conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 {micro}M binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1and UGGT2 activity at concentrations higher than 750 {micro}M in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N -glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N -glycan. Significance StatementWhen a candidate drug target is the product of a housekeeping gene - i.e. it is important for the normal functioning of the healthy cell - availability of inhibitors for tests and assays is of paramount importance. One such housekeeping protein is UGGT, the enzyme that makes sure that only correctly folded glycoproteins can leave the endoplasmic reticulum for further trafficking through the secretory pathway. UGGT is a potential drug target against viruses, in certain instances of congenital rare disease, and against some cancers, but no UGGT inhibitors are known yet. We discovered and describe here a small molecule that binds human UGGT1 in vitro and inhibits both isoforms of human UGGT in cellula. The compound paves the way to testing of UGGT inhibition as a potential pharmacological strategy in a number of medical contexts.

biochemistry↗

bioRxiv2021

MotSASi: Functional Short Linear Motifs (SLiMs) prediction based on genomic single nucleotide variants and structural data

Short linear motifs (SLiMs) are key to cell physiology mediating reversible protein-protein interactions. Precise identification of SLiMs remains a challenge, being the main drawback of most bioinformatic prediction tools their low specificity (high number of false positives). An important, usually overlooked, aspect is the relation between SLiMs mutations and disease. The presence of variants in each residue position can be used to assess the relevance of the corresponding residue(s) for protein function, and its (in)tolerance to change. In the present work, we combined sequence variant information and structural analysis of the energetic impact of single amino acid substitution (SAS) in SLiM-Receptor complex structure, and showed that it significantly improves prediction of true functional SLiMs. Our strategy is based on building a SAS tolerance matrix that shows, for each position, whether one of the possible 19 SAS is tolerated or not. Herein we present the MotSASi strategy and analyze in detail 4 SLiMs involved in intracellular protein trafficking. Our results show that inclusion of variant and sequence information significantly improves both prediction of true SLiMs and rejection of false positives, while also allowing better classification of variants inside SLiMs, a results with a direct impact in clinical genomics.

molecular biology↗

bioRxiv2019

Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP glucose:glycoprotein glucosyl-transferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecules flexibility. Principal component analysis of the MD trajectories affords a description of UGGTs overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These inter-domain motions modify the accessible surface area of the enzymes central saddle, likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name "Parodi limit" the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 [A]. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a "one-size-fits-all adjustable spanner" substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzymes active site.

biochemistry↗

bioRxiv2018

VarQ: a tool for the structural analysis of Human Protein Variants

Understanding the functional effect of Single Amino acid Substitutions (SAS), derived from the occurrence of single nucleotide variants (SNVs), and their relation to disease development is a major issue in clinical genomics. Even though there are several bioinformatic algorithms and servers that predict if a SAS can be pathogenic or not they give little or non-information on the actual effect on the protein function. Moreover, many of these algorithms are able to predict an effect that no necessarily translates directly into pathogenicity. VarQ Web Server is an online tool that given an UniProt id automatically analyzes known and user provided SAS for their effect on protein activity, folding, aggregation and protein interactions among others. VarQ assessment was performed over a set of previously manually curated variants, showing its ability to correctly predict the phenotypic outcome and its underlying cause. This resource is available online at http://varq.qb.fcen.uba.ar/.\n\nContact: lradusky@qb.fcen.uba.ar\n\nSupporting Information & Tutorials may be found in the webpage of the tool.

bioinformatics↗