Biology subjects

Gallardo, P.

Publications and source records attributed to Gallardo, P..

10 recordsLinked to original sources

bioRxiv2026

The chaperone Jjj2 regulates nucleoporin condensation in budding yeast

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport through a selective permeability barrier established by nucleoporins (Nups) that contain intrinsically disordered phenylalanine-glycine (FG) repeats. Due to these domains, FG-Nups are prone to condensation, with a potential to transition into insoluble aggregates. How cells keep FG-Nups in a soluble, functional state during NPC assembly and within the native NPC remains poorly understood. Here, we identify the uncharacterized yeast J-domain protein Jjj2 as a Nup chaperone. Disrupting Jjj2 function or its interaction with Hsp70 chaperones triggers the accumulation of newly synthesized Nups in cytoplasmic condensates. Conversely, Jjj2 overexpression suppresses Nup condensation but also disrupts the NPC permeability barrier and is highly toxic. Overall, our data show that Jjj2, in concert with Hsp70, controls Nup phase state. This activity must be tightly regulated; while Jjj2 prevents condensation of newly produced Nups, its overactivity compromises nucleocytoplasmic compartmentalization.

cell biology↗

bioRxiv2025

Conserved intrinsically disordered region of DNAJB6 dictates its surveillance of FG-Nup condensates

Molecular chaperones are known for their role in preventing protein aggregation and assisting proteins in reaching their structurally functional state. DNAJB6, a J-domain protein that partners with Hsp70s and nucleotide exchange factors, is very potent in preventing amyloid formation of proteins with large intrinsically disordered regions (IDRs), including several disease-associated proteins. Complementary to this, we recently demonstrated a role for DNAJB6 in surveilling FG-Nucleoporins (FG-Nups) phase transitions and highlighted its role in nuclear pore complex assembly. We expand on this by showing that this activity of phase state surveillance is directed to several FG-Nups and shared with the closely related DNAJB2 and DNAJB8. We demonstrate that the surveillance mechanism of DNAJB6 is encoded in an unusually highly conserved IDR that promotes the formation of stable, gel-like assemblies of the chaperone itself. These assemblies likely provide a stable environment that can outcompete stable homotypic FG-Nup interactions and instead favors multivalent heterotypic chaperone:FG-Nup interactions. The evolutionary conservation of the DNAJB6-IDR, mutant analyses from both experimental in vitro and in cell data, and multiscale molecular dynamics simulations suggest that the sequence space for encoding stable gel-like assemblies is narrow and optimized to avoid self-aggregation while providing potent anti-amyloidogenic capacity.

biochemistry↗

bioRxiv2025

Soybean aphids exploit abscisic acid signaling to suppress jasmonate defense responses

SummaryO_LISoybean aphids (Aphis glycines) can induce susceptibility on soybean (Glycine max) during colonization. However, the mechanism for this process is not known. Based on previous transcriptome analyses, we hypothesized that aphids block effective jasmonate (JA) defenses by inducing an antagonistic abscisic acid (ABA) signal. C_LIO_LITo test this hypothesis, we used a combination of gene expression analyses, measurements of hormone levels, and aphid bioassays on plants with reduced expression of ABA-related genes. C_LIO_LIAphid feeding attenuated JA responses in soybean plants and facilitated the growth of a chewing herbivore. Aphid-treated plants had increased levels of cis-JA but not biologically active JA-isoleucine, and aphid feeding induced expression of genes associated with JA-Ile catabolism. In parallel, aphid-feeding induced higher levels of ABA. ABA treatment and knockdown lines impaired in ABA biosynthesis (aba2-RNAi) or signaling (scof-1-RNAi), showed that ABA suppressed wound-induced JA responses. Aphid populations were significantly reduced on ABA-deficient plants and aphid-regulated attenuation of JA signaling was abolished in these lines. Remarkably, plants defective in ABA signaling had increased JA signaling in the absence of stressors. C_LIO_LIOur results indicate that, in soybean, the ABA pathway is necessary to control basal levels of JA and soybean aphids exploit this ABA-JA antagonism to suppress plant defenses. C_LI

plant biology↗

bioRxiv2024

Imaging-Based Quantitative Assessment of Biomolecular Condensates in vitro and in Cells

The formation of biomolecular condensates contributes to intracellular compartmentalization, and plays an important role in many cellular processes. The characterization of condensates is however challenging, requiring advanced biophysical or biochemical methods that are often less suitable for in vivo studies. A particular need for easily accessible yet thorough methods that enable the characterization of condensates across different experimental systems thus remains. To address this, we present PhaseMetrics, a semi-automated FIJI-based image analysis pipeline tailored for quantifying particle properties from microscopy data. Tested using the FG-domain of yeast nucleoporin Nup100, PhaseMetrics accurately assesses particle properties across diverse experimental setups, including in vitro, Xenopus egg extracts, and cellular systems. It reliably detects changes induced by various conditions such as the presence of polyethylene glycol, 1,6-hexanediol, a salt gradient, and the molecular chaperone DNAJB6b. By enabling the accurate representation of the variability within the population and the detection of subtle changes at the single particle level, the method complements conventional biochemical assays. Combined, PhaseMetrics is an easily accessible, customizable pipeline that enables imaging-based quantitative assessment of biomolecular condensates in vitro and in cells, providing a valuable addition to the current toolbox.

biochemistry↗

bioRxiv2024

Evaluation of Octopus maya enzyme activity of the digestive gland and gastric juice

BackgroundAs the demand for Octopus maya grows, sustainable farming practices become essential to prevent overexploitation. Thus, its farming development can be a sustainable alternative to traditional fishing. Understanding the digestive dynamics is essential for devising optimal dietary formulations in aquaculture, particularly the role of enzymes like cathepsins and others. Despite the progress in understanding cephalopod digestion, little is known about the specific functioning of the digestive enzymes responsible for breaking down protein substrates. This knowledge gap underscores the need for further research to ensure O. maya population sustainable management. Methods and ResultsDietary formulations are identified for cephalopods by characterizing O. maya digestive enzymes present in the digestive gland and gastric juice. The present investigation revealed that acidic proteases showed a peak activity at higher temperatures than alkaline proteases. Inhibitors confirmed the presence of H, L, and D cathepsins. Noteworthy is a lower activation energy of alkaline enzymes compared to acidic, ones highlighting an intriguing aspect of O. mayas digestive physiology. ConclusionOverall, this research provides valuable insights into O. maya digestive enzyme functions representing a significant advancement in formulating diets crucial for octopus successful farming that may help to fully understand its physiology.

physiology↗

bioRxiv2023

Dynamic molecular mechanism of the nuclear pore complex permeability barrier

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.

biophysics↗

bioRxiv2023

Nucleoporin Nsp1 surveils the phase state of FG-Nups

Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis. HighlightsO_LINups form native cytosolic condensates C_LIO_LINsp1 reduction mimics NPC aging phenotypes C_LIO_LINsp1 acts as phase state modulator of FG-Nups C_LIO_LINsp1 shares surveillance function with classical chaperones C_LI

cell biology↗

bioRxiv2021

The molecular chaperone DNAJB6 provides surveillance of FG-Nups and is required for interphase nuclear pore complex biogenesis

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage/crossing of biomolecules. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separate1,2 and aggregate when isolated3. It has remained largely unclear how FG-Nups are protected from making inappropriate interactions during NPC biogenesis. We found that DNAJB6, a molecular chaperone of the heat shock protein network, formed foci next to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Reversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and they could be identified as herniations at the nuclear envelope (NE). Immunoelectron tomography showed that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Interestingly, loss of DNAJB6 results in annulate lamellae, which are structures containing partly assembled NPCs associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG-region of several FG-Nups in cells and in vitro. Together, our data show that DNAJB6 provides quality control during NPC biogenesis and is the first molecular chaperone that is involved in the surveillance of native intrinsically disordered proteins, including FG-Nups.

molecular biology↗

bioRxiv2021

Landscapes and bacterial signatures of mucosa-associated intestinal microbiota in Chilean and Spanish patients with inflammatory bowel disease

Inflammatory bowel diseases (IBD), which include ulcerative colitis (UC) and Crohns disease (CD), cause chronic inflammation of the gut, affecting millions of people worldwide. IBD have been frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is generally characterized by an increase in abundance of Proteobacteria such as Escherichia coli, and a decrease in abundance of Firmicutes such as Faecalibacterium prausnitzii (an indicator of a healthy colonic microbiota). The mechanisms behind the development of the IBD and the dysbiosis are incompletely understood. Using samples from colonic biopsies, we studied the mucosa-associated intestinal microbiota in Chilean and Spanish patients with IBD. In agreement with previous studies, microbiome comparison between IBD patients and non-IBD controls indicated that dysbiosis in these patients is characterized by an increase of pro-inflammatory bacteria (mostly Proteobacteria) and a decrease of commensal beneficial bacteria (mostly Firmicutes). Notably, bacteria typically residing on the mucosa of healthy individuals were mostly obligate anaerobes, whereas in the inflamed mucosa an increase of facultative anaerobe and aerobic bacteria was observed. We also identify potential co-occurring and mutually exclusive interactions between bacteria associated with the healthy and inflamed mucosa, which appear to be determined by the oxygen availability and the type of respiration. Finally, we identify a panel of bacterial biomarkers that allow the discrimination between eubiosis from dysbiosis with a high diagnostic performance (96% accurately), which could be used for the development of non-invasive diagnostic methods. Thus, this study is a step forward toward understanding the landscapes and alterations of mucosa-associated intestinal microbiota in patients with IBD.

microbiology↗

bioRxiv2019

Best-compromise nutritional menus for childhood obesity

Childhood obesity is an undeniable reality and has shown a rapid growth in many countries. Obesity at an early age not only increases the risks of chronic diseases but also produces a problem for the whole healthcare system. One way to alleviate this problem is to provide each patient with an appropriate menu that can be defined with a mathematical model. Existing mathematical models only partially address the objective and constraints of childhood obesity; therefore, the solutions provided are insufficient for health specialists to prepare nutritional menus for individual patients. This manuscript proposes a multiobjective mathematical programming model to aid healthy nutritional menu planning to prevent childhood obesity. This model enables a plan for combinations and amounts of food across different schedules and daily meals. This approach minimizes the major risk factors of childhood obesity (i.e., glycemic load and cholesterol intake). In addition, it considers the minimization of nutritional mismatch and total cost. The model is solved using a deterministic method and two metaheuristic methods. To complete this numerical study, test instances associated with children aged 4-18 years old were created. The quality of the solutions generated using the three methods was similar, but the metaheuristic methods provided solutions in less computational time. The numerical results indicate proper guidelines for personalized plans for individual children.

scientific communication and education↗