Biology subjects

Oroz, J.

Publications and source records attributed to Oroz, J..

7 recordsLinked to original sources

bioRxiv2026

Human NLRP1 and NLRP3 interact driving inflammation in inflammosomopathies and inflammatory diseases

Inflammasomes are multiprotein complexes that form and activate after exposure to pathogenic microbes and host danger signals that trigger an inflammatory response. Although NLRP1 and NLRP3 inflammasomes share structural similarities and can be activated by similar stimuli, no evidence of heterotypic inflammasome assemblies has been reported. Here, we identify a unique interaction between NLRP1 and NLRP3 in human cells, forming a hybrid inflammasome, to drive inflammation. NLRP1 is essential for this hydrid inflammasome activation and NLRP3-mediated Caspase-1 activation and release of IL-1{beta} and IL-18. The presence of the heterocomplex inflammasome was confirmed in blood samples from patients after kidney transplantation and is associated with inflammatory responses driven by NLRP3 and MEFV mutations that cause inflammasomopathies. Our findings reveal an unexpected level of intricacy in inflammasome composition, pinpointing hybrid targets that may pave the way for innovative pharmacological treatments for inflammatory disorders. Significance StatementPrevious findings showed interactions between NLRP3 and NLRC4 or NLRP3 and NLRP11 showing that would be possible the interaction of the inflammasomes as supercomplexes and not working alone. Now, we show a new inflammasome heterocomplex inflammasome between NLRP1 and NLRP3 which is associated to the inflammatory profile in autoimmune diseases patietns and transplanted patients. These findings could open a new research topic for the design of dual inflammasomes inhibitors.

immunology↗

bioRxiv2025

Pathological mutation in SMN impairs modulation of GAR1 phase separation linking condensate dysfunction to Spinal Muscular Atrophy

Pathological mutations in liquid-liquid phase separation (LLPS) scaffold proteins have been linked to biomolecular condensate dysfunction in neurodegenerative diseases, while the possible impact of client protein mutations remains unclear. In spinal muscular atrophy (SMA), the disease-associated E134K mutation in the Tudor domain (TD) of the survival motor neuron (SMN) protein disrupts its interaction with GAR1, an RGG-rich protein within the H/ACA small nucleolar ribonucleoprotein complex. The consequences of this impaired interaction have not been elucidated. Here, we identified GAR1 as an LLPS scaffold protein that is phase separated in nuclear compartments and forms gel-like condensates via complex coacervation with RNA in vitro. In cells, we reveal that SMN co-localizes with GAR1 in Cajal bodies and modulates its dynamics. Using confocal microscopy and NMR spectroscopy, we further show that SMN TD is a client protein that regulates the architecture and dynamics of GAR1 condensates through competitive RNA interactions, implicating GAR1 phase separation in RNA accumulation and release processes regulated by SMN. Notably, the SMA-associated E134K variant of SMN exhibits a reduced affinity for GAR1, impairing the modulation of GAR1 condensates and displacement RNA. Our findings suggest a mechanistic link between phase separation dysregulation and SMA, driven by disrupted scaffold-client interactions, that highlights the therapeutic potential of targeting SMN-dependent condensate regulation in SMA.

biochemistry↗

bioRxiv2022

NEK7 activates the NLRP1 Inflammasome

Inflammasomes including those assembled by NLRP1 and NLRP3 regulate the innate immune system by inducing interleukin (IL)-1{beta} and IL-18 maturation. Inflammasomes are functionally regulated by post-translational modifications such as phosphorylation. The current paradigm posits that NEK7 is the essential and seletive activator of NLRP3; whether this kinase interacts with NLRP3 structurally-related member, NLRP1, has never been explored. Here, we find that NEK7 binds to NLRP1 and promotes its activation independently of NLRP3. IL-1{beta} maturation induced by NLRP1 or NLRP3 inflammasome activators, but not those of the NLRC4 or AIM2 inflammasome is impared in Nek7 deficient cells. This discovery expands the spectrum of NEK7 actions in the regulation of inflammasome pathways.

immunology↗

bioRxiv2022

Fused in Sarcoma undergoes cold denaturation: implications on phase separation

The mediation of fused in sarcoma (FUS) protein liquid-liquid phase separation (LLPS) is generally attributed to the low-complexity and disordered domains, while the role of its folded domains remains unknown. In this work we questioned the role of the folded domains on the full-length (FL) FUS LLPS and studied the influence of several metabolites, ions and overall conditions on the LLPS process using turbidity assays, differential interference contrast microscopy and nuclear magnetic resonance spectroscopy. We demonstrate that FL FUS LLPS is highly responsive to the surrounding conditions, and that overall intrinsic disorder is crucial for LLPS. To promote such disorder, we reveal that the FUS RNA-recognition domain (RRM) and the zinc-finger motif (ZnF) undergo cold denaturation above 0{o}C, at a temperature that is determined by the conformational stability of the ZnF domain. We hypothesize that, in cold shock conditions, cold denaturation might provide a pathway that exposes additional residues to promote FUS self-assembly. Such findings mark the first evidence that FUS globular domains may have an active role in stress granule formation in cold stress.

biophysics↗

bioRxiv2020

Glycine Rich Segments Adopt Polyproline II Helices Which May Contribute to Biomolecular Condensate Formation

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Considering NMR studies of the Gly-rich H. harveyi "snow flea" antifreeze protein, we recently proposed that Gly-rich sequences, such as the third "RGG" region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. In cells, the polyproline II population of these peptides could be augmented by binding profilin protein or SH3, WW or OCRE domains, association with RNA or assembly into polyproline II helical bundles. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.

biophysics↗

bioRxiv2019

Conformational Priming of RepA-WH1 for Functional Amyloid Conversion Detected by NMR Spectroscopy

How proteins with a stable globular fold acquire the amyloid state is still largely unknown. RepA is a versatile plasmidic DNA binding protein, functional either as a transcriptional repressor or as an initiator or inhibitor of DNA replication, the latter through the assembly of an amyloidogenic oligomer. Its N-terminal domain (WH1) is responsible for discrimination between these functional abilities by undergoing hitherto unknown structural changes. Furthermore, when expressed alone, RepA-WH1 behaves as a synthetic prion-like protein causing an amyloid proteinopathy in bacteria. RepA-WH1 is a stable dimer whose conformational dynamics had not been explored. Here we have studied it through NMR {1H}-15N relaxation and H/D exchange kinetics measurements. The N- and the C- terminal -helices, which lock the WH1 fold in each subunit of the dimer, as well as an internal amyloidogenic loop, show reduced stability and are partially unfolded in solution. S4-indigo, a small molecule ligand known to interfere with the amyloid assembly of RepA-WH1, binds to and tethers the N-terminal -helix and a {beta}-hairpin that is involved in dimerization, thus providing evidence for a priming role of fraying ends and dimerization switches in the amyloidogenesis of folded proteins.

biochemistry↗

bioRxiv2019

Dynamic Aha1 Co-Chaperone Binding to Human Hsp90

Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. Because of the inherent low ATPase activity of human Hsp90, the co-chaperone Aha1, which is the only known ATPase stimulator in eukaryotes, is important for regulation of Hsp90s allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 full-length complex. Here, we characterize the solution structure of unmodified human Hsp90 in complex with Aha1 using NMR spectroscopy. We show that the 214 kDa complex adopts multiple conformations in the absence of nucleotide. Interaction with Aha1 induces structural changes near the nucleotide-binding site in Hsp90s N-terminal domain, providing a basis for its ATPase-enhancing activity. Moreover, the E67K mutation in Aha1 strongly diminishes the interaction, supporting a two-step binding mechanism. Our data reveal important aspects of this pivotal chaperone/co-chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.

biophysics↗