Margarita Menéndez

Coupling between the chaperone and degradation systems, particularly under stress, is essential for eliminating unfolded proteins. The co-chaperone Bag1 links Hsp70 to the 26 S proteasome, recruiting Hsp70-bound clients for proteasomal degradation. Here, we present cryo–electron microscopy structures of the Bag1-bound 26 S proteasome, revealing unprecedented conformational rearrangements within the 19 S regulatory particle. Bag1 binding to the Rpn1 induces a marked reconfiguration of AAA ⁺ adenosine triphosphatase (ATPase) ring, disrupting its canonical spiral staircase and remodeling the central channel architecture. This reconfiguration generates a large cavity above the substrate entry gate of the 20 S core particle. The conserved pore-2 loops of ATPases Rpt2 and Rpt5 play critical roles in opening of the 20 S gate, enabling substrate entry into proteolytic chamber independently of ubiquitination. These findings suggest a previously unknown mechanism of the proteasomal degradation, by which remodeling the central cavity and 20 S gate in the presence of Bag1, possibly bypassing the need for ubiquitination.

Wetlands, widely distributed and hotspots of biodiversity, play a crucial role in global biogeochemical cycles and human well-being. However, despite their ecological importance, wetlands worldwide are under threat due to widespread conversion into agricultural fields, leading to changes in hydrology, increased salinity, and more frequent eutrophication. In response to these challenges, constructed wetlands are created to treat agricultural wastewater and mitigate eutrophication. This study aims to assess the effect of natural vs. constructed wetlands on ecosystem functioning (organic matter decomposition of the dominant vegetation: Phragmites australis and Typha angustifolia). We conducted this study in the Ebro River Delta (NE Spain), which represents a deltaic wetland affected by agricultural land-use changes, examining two constructed and two natural wetlands. Our findings indicate that the influence of agricultural runoff on the decomposition process was similar in both types of wetlands, suggesting that freshwater agricultural runoff has a consistent effect on ecosystem functioning, regardless of its origin, natural vs. constructed. Differences in macroinvertebrate communities associated with leaf litter were likely due to specific conductivity but did not impact decomposition rates. The estimated time required to decompose 95% of the T. angustifolia litter produced annually in the studied wetlands ranged from 288 to 856 days. In constructed wetland, this decomposition time exceeded one year, contributing to soil formation and carbon sequestration in wetland ecosystems. Our study suggests that the utilization of constructed wetlands for treating agricultural runoff can aid in mitigating the impacts of agricultural land use in these areas.

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host–pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 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 that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC₅ ^DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccC^DUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC₅ ^DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC₅ ^DUF with ATP/Mg²⁺, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC₅ ^DUF, it is hypothesized that MtbEccC₅ ^DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.

Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well‐characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single‐molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau‐RD_244‐368). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro‐amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti‐amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau‐RD_244‐368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.

Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well‐characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single‐molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau‐RD_244‐368). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro‐amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti‐amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau‐RD_244‐368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.

The Neuronal Calcium Sensor 1 and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα 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 Gα 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 Ca ²⁺ signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα 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 Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca ²⁺ 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.

We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α₃β₆-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a β-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation.

Lipid nanocarriers, such as niosomes, are considered attractive candidates for non-viral gene delivery due to their suitable biocompatibility and high versatility. In this work, we studied the influence of incorporating chloroquine in niosomes biophysical performance, as well as the effect of non-ionic surfactant composition and protocol of incorporation in their biophysical performance. An exhaustive comparative evaluation of three niosome formulations differing in these parameters was performed, which included the analysis of their thermal stability, rheological behavior, mean particle size, dispersity, zeta potential, morphology, membrane packing capacity, affinity to bind DNA, ability to release and protect the genetic material, buffering capacity and ability to escape from artificially synthesized lysosomes. Finally, in vitro biological studies were, also, performed in order to determine the compatibility of the formulations with biological systems, their transfection efficiency and transgene expression. Results revealed that the incorporation of chloroquine in niosome formulations improved their biophysical properties and the transfection efficiency, while the substitution of one of the non-ionic surfactants and the phase of addition resulted in less biophysical variations. Of note, the present work provides several biophysical parameters and characterization strategies that could be used as gold standard for gene therapy nanosystems evaluation.