Biology subjects

Menendez, M.

Publications and source records attributed to Menendez, M..

6 recordsLinked to original sources

bioRxiv2025

Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

The 26S proteasome primarily degrades proteins marked by polyubiquitin chains. Although ubiquitin-independent pathways for proteasomal degradation exist, the mechanisms involved remain poorly understood. Bag1 links the Hsp70 chaperone to the 26S proteasome, recruiting Hsp70-bound aberrant proteins for degradation. Here, we present high-resolution cryo-EM structures of the Bag1-bound 26S proteasome, which reveal unprecedented conformational changes within the 19S regulatory particle. Bag1 binding to the Rpn1 subunit induces a dramatic reconfiguration of AAA+ ATPase subunits, disrupting the canonical spiral staircase conformation and remodeling the central channel architecture. This creates a large cavity above the substrate entry gate of the 20S core particle, enabling the direct entry of Hsp70 clients into the proteolytic chamber. Thus, in this ubiquitin-independent degradation pathway, unfolded proteins can bypass the need for both client ubiquitination and ATP hydrolysis for degradation.

molecular biology↗

bioRxiv2024

New insights into the domain of unknown function DUF of EccC5, the pivotal ATPase providing the secretion driving force to the ESX5 secretion system

Type VII secretion (T7S) systems, also referred to as ESAT6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implication in cell homeostasis and host pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis and a pandemic accounting for more than 1 million deaths every year. The ESX5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors, the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase operating through four globular cytosolic domains, an N-terminal domain of unknown function (EccCDUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX3 and ESX5 systems have revealed EccCDUF as an ATPase-like fold domain with potential ATPase activity, and whose functionality is essential for secretion. Here we report the crystal structure of MtbEccC5DUF domain at 2.05 [A] resolution, which unveils a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP-binding and hydrolysis. Our crystallographic study, together with a biophysical assessment of MtbEccC5DUF interaction with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. We show that this degeneration is also present in DUF domains of other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, and based on an in-silico model of MtbEccC5 N-terminal region, we propose that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerise. Observations that call the attention on DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process.

microbiology↗

bioRxiv2022

The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A

The Neuronal Calcium Sensor 1 and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to G have revealed how Ric-8A phosphorylation promotes G recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with G subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with G. Our data show that the binding of NCS-1 and G to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a profound structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards G when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here that define the NCS-1/Ric-8A interface will allow the development of therapeutic synapse function regulators with improved activity and selectivity.

biochemistry↗

bioRxiv2020

Tau amyloidogenesis begins with a loss of its conformational polymorphism

Knowledge on the molecular bases of early amyloid assembly is fundamental to understand its structure-dysfunction relationship during disease progression. Tauopathies, a well-defined set of neurodegenerative disorders that includes Alzheimers disease, are characterized by the pathological amyloid aggregation of tau. However, the underlying molecular mechanisms that trigger tau aggregation and toxicity are poorly understood. Here, using a single-molecule approach, AFM-based single molecule-force spectroscopy (AFM-SMFS), combined with a protein-engineering mechanical protection strategy, we have analyzed the fluctuations of the conformational space of tau during the start of its pathological amyloid assembly. Specifically, we have analyzed the region that includes the four tau microtubule-binding repeats, known to play a key role on tau aggregation. We find that, unlike other amyloid-forming proteins, tau aggregation is accompanied by a decrease of conformational polymorphism, which is driven by amyloid-promoting factors, such as the {Delta}280K and P301L mutations, linked to Frontotemporal Dementia-17, or by specific chemical conditions. Such perturbations have distinct effects and lead to different tau (aggregate) structures. In addition to providing insight into how tau aggregates in a context dependent manner, these findings may help delve into how protein aggregation-based diseases, like Alzheimers, might be treated using monomer fluctuations as a pharmacological target. O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/158923v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@951f1borg.highwire.dtl.DTLVardef@2d5f5borg.highwire.dtl.DTLVardef@cf7776org.highwire.dtl.DTLVardef@abe671_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold

Functional amyloids are present in a wide variety of organisms ranging from bacteria to humans. Experience-dependent aggregation of the cytoplasmic polyadenylation element-binding (CPEB) prion-like protein to a translationally active state has emerged as a plausible biochemical substrate of long-lasting memories. CPEB aggregation is driven by prion-like domains (PLD) that are highly divergent in sequence across species. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD in vitro using single-molecule and bulk biophysical methods and compare them with those previously reported for neuronal Drosophila CPEB, Orb2 PLD. The existence of transient oligomers and mature filaments suggests similarities in the late stages of the assembly pathway for both PLDs. However, while prior to aggregation the Orb2 PLD monomer remains as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising -helical structures that evolve to coiled-coil species, suggesting structural differences at the beginning of their amyloid assembly pathways. Our results show how divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

neuroscience↗

bioRxiv2019

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry

Adenovirus minor coat protein VI contains a membrane-disrupting peptide which is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and non-infectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wildtype (Ad5-wt) particles. Cryo-EM difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating. Significance StatementCorrect assembly of an adenovirus infectious particle involves the highly regulated interaction of more than ten different proteins as well as the viral genome. Here we examine the interplay between two of these proteins: the major core protein VII, involved in genome condensation, and the multifunctional minor coat protein VI. Protein VI binds to the inner surface of adenovirus hexons (trimers of the major coat protein) and contains a lytic peptide which must be released during entry to ensure endosome rupture. We present data supporting a dynamic competition model between proteins VI and VII for hexon binding during assembly. This competition facilitates the release of the lytic peptide from the hexon cavity and ensures virus escape from the early endosome.

microbiology↗