Biology subjects

Carvalho, P.

Publications and source records attributed to Carvalho, P..

15 recordsLinked to original sources

bioRxiv2026

Selective quality control of mistargeted mitochondrial proteins at the endoplasmic reticulum

In eukaryotic cells, the function of each organelle depends on its unique protein composition. Protein targeting errors threaten organelle identity and function, yet how mistargeting errors are detected and resolved remains poorly understood. Here, we show that mitochondrial import stress drives widespread rerouting of mitochondrial proteins to the endoplasmic reticulum (ER), with strong enrichment for hydrophobic oxidative phosphorylation (OXPHOS) components. Using proximity proteomics, a split-fluorescence reporter system, and genome-wide CRISPR screening, we find that mistargeted proteins partition into distinct classes with divergent fates, ranging from stable ER residence to rapid degradation by ER-associated degradation (ERAD). The clearance of these mislocalized proteins involves partially redundant ERAD branches, with the ubiquitin ligase MARCHF6 playing a central role. Together, these findings establish the ER as a key organelle for handling mistargeted mitochondrial proteins and reveal how ER quality control maintains proteostasis during mitochondrial dysfunction.

cell biology↗

bioRxiv2026

Regulated conformational transitions in seipin define a functional ER–lipid droplet interface

Lipid droplets (LDs) are key organelles in cellular lipid homeostasis that form at the endoplasmic reticulum (ER) through a sequence of membrane rearrangements. While seipin emerged as an essential protein complex for LD biogenesis, how seipin-mediated LD formation proceeds beyond the initial step of neutral lipid nucleation remains unknown. Using a combination of in vitro and in-cell cryogenic electron microscopy (cryo-EM), ultrastructural expansion microscopy, molecular simulations and tailored genetic perturbations, we show that the seipin transmembrane domains undergo large-scale conformational rearrangements that define the architecture of the ER-LD interface and enable LD growth. Cryo-EM of purified Xenopus seipin revealed coexistence of two states: a compact "closed" conformation, consistent with early LD biogenesis, and an "open" conformation in which the transmembrane helices splay out laterally. Molecular dynamics simulations indicate that this open state induces local membrane curvature and promotes triacylglycerol accumulation. We identify conserved flexible linkers between the seipin luminal and transmembrane regions that act as mechanical hinges, enabling this conformational transition. We demonstrate that mutations in these hinge regions hinder seipin opening and affect LD formation in yeast and human cells. Analysis of native ER-LD contacts in human cells using light microscopy and cryo-electron tomography confirms that the seipin complex opens to establish stereotypical ~21-nm necks connecting the ER bilayer and LD monolayer. Moreover, we identify the liver-enriched microprotein SMLR1 as an inhibitor of this seipin conformational transition, providing a regulatory mechanism for seipin-dependent lipid storage in a tissue-specific manner. Together, these data establish seipin opening as a key structural rearrangement at the ER-LD interface that is essential for LD biogenesis and growth.

Cell Biology↗

bioRxiv2026

Lipid remodelling enables adaptation to chronic hyperosmotic stress

Lipid droplets (LDs) accumulate in response to diverse cellular stresses. However, their regulation and physiological roles remain poorly understood in most contexts. Here, we show that, in budding yeast, chronic hyperosmotic stress induces sustained LD accumulation. Unlike the transient LD response observed during acute osmotic shock, chronic stress triggers prolonged, Dga1-dependent triacylglycerol synthesis. In the absence of triacylglycerol synthesis cellular fitness is severely affected. Lipidomic profiling reveals extensive membrane remodelling during chronic hyperosmotic stress, most notably a shift from phosphatidylethanolamine to phosphatidylcholine. In LD-deficient cells, the stress-induced PC increase is blunted and manipulation of PC synthesis modulates the fitness of triacylglycerol-deficient cells under hyperosmotic stress. Thus, LD accumulation and phospholipid remodelling underlie an adaptive response to chronic hyperosmotic stress. SummaryThis work demonstrates that membrane remodelling occurs in cells experiencing chronic hyperosmotic stress. Both triacylglycerol and phosphatidylcholine levels are increased. Cell fitness depends upon increased triacylglycerol synthesis and is further modulated by manipulating phosphatidylcholine levels.

cell biology↗

bioRxiv2025

Organization of the Yeast Seipin Complex Reveals Differential Recruitment of Regulatory Proteins

Lipid droplets (LDs) are neutral lipid storage organelles that emerge from the endoplasmic reticulum (ER). Their assembly occurs in ER regions enriched with seipin which, through its homooligomeric ring-like structure, facilitates neutral lipid nucleation. In yeast, seipin (Sei1) partners with Ldb16, Ldo45 (yeast homologue of human LDAF1) and Ldo16, which regulate LD formation and consumption. How the molecular architecture of the yeast seipin complex and its interaction with regulatory proteins adapt to different metabolic conditions remains poorly understood. Here, we show that multiple Ldb16 regions contribute differently to recruiting Ldo45 and Ldo16 to the seipin complex. Using an in-vivo site-specific photo-crosslinking approach, we further show that Ldo45 resides at the center of the seipin ring both in the absence and presence of neutral lipids. Interestingly, neutral lipid synthesis leads to the recruitment of Ldo45 but not Ldo16 to the complex. Our findings suggest that the seipin complex serves as a pre-assembled scaffold for lipid storage that can be remodeled in response to increased neutral lipid availability.

cell biology↗

bioRxiv2025

Pex30-dependent membrane contacts sites maintain ER lipid homeostasis

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the Dysferlin domain (DysF) on Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo . The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that ER-Vacuole MCS requires Pex30 C-terminal Domain of Unknown Function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

cell biology↗

bioRxiv2024

LC-MS/MS characterization of SOBERANA(R)02, a receptor binding domain-tetanus toxoid conjugate vaccine against SARS-CoV-2

SOBERANA(R)02 is a safe and effective conjugate vaccine against SARS-CoV-2, produced using the maleimide-thiol chemistry. In this vaccine, the Cys538 in the recombinant receptor binding domain (RBD) of SARS-CoV-2 is linked, through a thiosuccinimide linker, to lysine residues of tetanus toxoid (TT) preparation. LC-MS/MS analysis revealed that TT is a complex mixture of proteins, similar to other TTs where the detoxified tetanus neurotoxin (d-TeNT) has been shown to be the most abundant protein (30-56%), regardless the quantification method used. The fifteen most abundant proteins account for approximately 78% of the total proteins. LC-MS/MS analysis of the activation process showed that 102 out of 107 lysine residues in the d-TeNT incorporated a maleimide group. In contrast, when tryptic peptides isolated by Ni2+-NTA affinity chromatography, were analyzed by LC-MS/MS, only 22 Lys residues in the d-TeNT were cross-linked to the RBD C-terminal tryptic peptide (538CVNF541-HHHHHH), probably due to steric hindrance. Twelve and eighteen conjugation sites were assigned based on the identification of linear peptides carrying a conjugated lysine residues ({Delta}m = +1454.58 Da or {Delta}m = +1472.59 Da) and cross-linked peptides with stabilized linker forms, respectively. Eight conjugation sites were coincidently assigned by both strategies. The assignment of the conjugation sites was manually validated by observed regularities (z[≥]3+, XIC, immonium ions, specific linker fragment ions) not considered by the identification software (PEAKS, Kojak and pLink2). The RBD was also conjugated, but to a lesser extent, to ten other low abundance carrier proteins. To our knowledge, this work is the first report of conjugation site assignment in a TT-based conjugate vaccine.

biochemistry↗

bioRxiv2024

OsbHLH089 and OsbHLH094 Modulate OsSLR1 Levels to Maintain Male Reproductive Fitness in Rice

DELLA proteins are a unique class of transcriptional regulators in plants, playing critical roles in diverse biological processes. Far from being only negative regulators of the Gibberellin (GA) signalling pathway, DELLAs act as central signalling hubs due to their versatile binding capacity and responsiveness to GA fluctuations. This adaptability allows DELLAs to interact with a wide array of proteins but also makes their functional study challenging: disruptions in DELLA function lead to pleiotropic effects across multiple pathways. To address this complexity, understanding DELLA interactors provides valuable insights into DELLAs nuanced functions and regulation. Building on this approach, we investigated novel OsSLR1 interactors, OsbHLH089 and OsbHLH094, members of the basic-helix-loop-helix transcription factor family. Our findings reveal that OsbHLH089 and OsbHLH094 are expressed in mature pollen grains, with the Osbhlh089/94 double mutant displaying smaller, indehiscent anthers and non-viable pollen, indicating their redundant role in pollen development during its late stages. Through ChIP-Seq and RNA-Seq analyses, we identified five target genes repressed by OsbHLH089 and OsbHLH094 (OsTDL1A, OsSPS1, OsDGD2{beta}, OspPGM, and OsDPE2) that are essential for pollen and anther development. Interestingly, we also observed the binding of these TFs to the OsSLR1 promoter. Notably, while other target genes were repressed, OsSLR1 was induced, with a significant protein accumulation in the double mutant compared to the Kitaake background in the late stages of pollen development. This suggests that OsSLR1 accumulation may compromise pollen viability, further highlighting the critical regulatory role of OsbHLH089 and OsbHLH094 in repressing OsSLR1 levels for proper pollen and anther development.

plant biology↗

bioRxiv2024

Suppression of TGF-β/SMAD signaling by an inner nuclear membrane phosphatase complex

Cytokines of the TGF-{beta} superfamily control essential cell fate decisions via receptor regulated SMAD (R-SMAD) transcription factors. Ligand-induced R-SMAD phosphorylation in the cytosol triggers their activation and nuclear accumulation. We determined how R-SMADs are inactivated by dephosphorylation in the cell nucleus to counteract signaling by TGF-{beta} superfamily ligands. We showed that R-SMAD dephosphorylation is mediated by an inner nuclear membrane associated complex containing the scaffold protein MAN1 and the CTDNEP1-NEP1R1 phosphatase. Structural prediction, domain mapping and mutagenesis revealed that MAN1 binds independently to the CTDNEP1-NEP1R1 phosphatase and R-SMADs to promote their inactivation by dephosphorylation. Disruption of this complex led to nuclear accumulation of R-SMADs and aberrant signaling, even in the absence of TGF-{beta} ligands. These findings establish CTDNEP1-NEP1R1 as the elusive R-SMAD phosphatase and reveal the mechanistic basis for TGF-{beta} signaling inactivation and how this process is disrupted by disease-associated MAN1 mutations.

cell biology↗

bioRxiv2023

Concurrent stimulation of diflufenican biodegradation and changes in the active microbiome in gravel revealed by Total RNA

The use of slowly degrading pesticides poses a particular problem when these are applied to urban areas such as gravel paths. The urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. We, therefore, endeavoured to stimulate the degradation of the pesticide diflufenican added to an urban gravel microcosm amended with dry alfalfa to increase microbial activity. In the present study, the formation of the primary diflufenican metabolite 2-[3-(Trifluoromethyl)phenoxy]nicotinic acid (commonly abbreviated as AE-B) was stimulated by the alfalfa amendment. The concurrent changes of the active microbial communities within the gravel were explored using shotgun metatranscriptomic sequencing of ribosomal RNA and messenger RNA. Our results showed, that while the active microbial communities in the gravel were dominated by bacteria with a relative abundance of 87.0 - 98.5 %, the eukaryotic groups, fungi and micro-eukaryotes, both had a 4-5 fold increase in relative abundance over time in the alfalfa amended treatment. Specifically, the relative abundance of microorganisms involved in degradation of complex carbon sources, Bacteroidetes, Verrucomicrobia, Sordariomycetes, Mortierellales, and Tremellales, were shown to increase in the alfalfa amended treatment. Further, the functional gene profile showed an increase in genes involved in increased activity and production of new biomass in the alfalfa treatment compared to the control, as well as pointing to genes potentially involved in biodegradation of complex carbon sources and the biotransformation of diflufenican.

microbiology↗

bioRxiv2022

A biology-inspired recurrent oscillator network for computations in high-dimensional state space

The dynamics of neuronal systems are characterized by hallmark features such as oscillations and synchrony. However, it has remained unclear whether these characteristics are epiphenomena or are exploited for computation. Due to the challenge of specifically interfering with oscillatory network dynamics in neuronal systems, we simulated recurrent networks (RNNs) of damped harmonic oscillators in which oscillatory activity is enforced in each node, a choice well-supported by experimental findings. When trained on standard pattern recognition tasks, these harmonic oscillator networks (HORNs) outperformed non-oscillatory architectures with respect to learning speed, noise tolerance, and parameter efficiency. HORNs also reproduced a substantial number of characteristic features of neuronal systems such as the cerebral cortex and the hippocampus. In trained HORNs, stimulus-induced interference patterns holistically represent the result of comparing sensory evidence with priors stored in recurrent connection weights, and learning-induced weight changes are compatible with Hebbian principles. Implementing additional features characteristic of natural networks, such as heterogeneous oscillation frequencies, inhomogeneous conduction delays, and network modularity, further enhanced HORN performance without requiring additional parameters. Taken together, our model allows us to give plausible a posteriori explanations for features of natural networks whose computational role has remained elusive. We conclude that neuronal systems are likely to exploit the unique dynamics of recurrent oscillators networks whose computational superiority critically depends on the oscillatory patterning of their nodal dynamics. Implementing the proposed computational principles in analog hardware is expected to enable the design of highly energy-efficient and self-adapting devices that could ideally complement existing digital technologies.

neuroscience↗

bioRxiv2022

Estimating sequence diversity of prion protein gene (PRNP) in Portuguese populations of two cervid species: red deer and fallow deer.

Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease (CWD) in cervids is now a rising concern in wildlife within Europe, after the detection of the first case in Norway in 2016, in a wild reindeer and until June 2022 a total of 34 cases were described in Norway, Sweden and Finland. The definite diagnosis is postmortem, performed in target areas of the brain and lymph nodes. Samples are first screened using a rapid test and, if positive, confirmed by immunohistochemistry and Western immunoblotting. The study of the genetics of the prion protein gene, PRNP, has been proved to be a valuable tool for determining the relative susceptibility to TSEs. In the present study, the exon 3 of PRNP gene of 143 samples from red deer (Cervus elaphus) and fallow deer (Dama dama) of Portugal was analyzed. Three single nucleotide polymorphisms (SNPs) were found in red deer - codon A136A, codon T98A, codon Q226E - and no sequence variation was detected in fallow deer. The low genetic diversity found in our samples is compatible with previous studies in Europe. The comparison with results from North America, suggests that the free-ranging deer from our study may present susceptibility to CWD, although lack of experimental data and the necessity of extensive survey are necessary to evaluate these populations.

genetics↗

bioRxiv2022

Regulated Degradation of the Inner Nuclear Membrane Protein SUN2 Maintains Nuclear Envelope Architecture and Function

Nuclear architecture and functions depend on dynamic interactions between nuclear components (such as chromatin) and inner nuclear membrane (INM) proteins. Mutations in INM proteins interfering with these interactions result in disease. However, mechanisms controlling the levels and turnover of INM proteins remain unknown. Here, we describe a mechanism of regulated degradation of the INM SUN domain-containing protein 2 (SUN2). We show that Casein Kinase II and the C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) have opposing effects on SUN2 levels by regulating SUN2 binding to the ubiquitin ligase Skp/Cullin1/F-Box{beta}TrCP (SCF{beta}TrCP). Upon binding to phosphorylated SUN2, SCF{beta}TrCP promotes its ubiquitination. Ubiquitinated SUN2 is membrane extracted by the AAA ATPase p97 and delivered to the proteasome for degradation. Importantly, accumulation of non-degradable SUN2 results in aberrant nuclear architecture, vulnerability to DNA damage and increased lagging chromosomes in mitosis. These findings uncover a central role of proteolysis in INM protein homeostasis.

cell biology↗

bioRxiv2021

Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosiss pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

biochemistry↗

bioRxiv2020

The derlin Dfm1 promotes retrotranslocation of folded protein domains from the endoplasmic reticulum

Endoplasmic reticulum (ER) proteins are degraded by proteasomes in the cytosol through ER-associated degradation (ERAD). This process involves retrotranslocation of substrates across the ER membrane, their ubiquitination and subsequent membrane extraction by the Cdc48/Npl4/Ufd1 ATPase complex prior delivery to proteasomes for degradation. Recently a mechanism for the retrotranslocation of misfolded substrates by the Hrd1 ubiquitin ligase complex was described. However, how substrates with folded luminal domains are retrotranslocated remains unknown. Here, we identify Dfm1 as an essential membrane component for the retrotranslocation of proteins with folded luminal domains. Both Dfm1 intramembrane rhomboid-like and the cytosolic Cdc48-binding domains are essential for substrate retrotranslocation. Substrate processing by Dfm1 and Cdc48 complex requires the ubiquitin shuttle factors Rad23/Dsk2 and the multi-ubiquitination enzyme Ufd2. Our findings suggest a pathway in which a series of ubiquitin modifying factors recruit Dfm1 to resolve a stalled retrotranslocation intermediate due to the presence of a folded luminal domain.

cell biology↗

bioRxiv2020

First description of a herpesvirus infection in genus Lepus

During the necropsies of Iberian hares obtained in 2018/2019, along with signs of the nodular form of myxomatosis, other unexpected external lesions were also observed. Histopathology revealed nuclear inclusion bodies in stromal cells suggesting the additional presence of a nuclear replicating virus. Transmission electron microscopy further demonstrated the presence of herpesvirus particles in the tissues of affected hares. We confirmed the presence of herpesvirus in 13 MYXV-positive hares by PCR and sequencing analysis. Herpesvirus-DNA was also detected in seven healthy hares, suggesting its asymptomatic circulation. Phylogenetic analysis based on DNA polymerase gene, grouped this virus within the gammaherpesviruses, close to herpesviruses from bats and rodents. We propose to name this new virus Leporid gammaherpesvirus 5 (LeHV-5), according to the International Committee on Taxonomy of Viruses standards. The impact of herpesvirus infection on the reproduction and mortality of the Iberian hare is yet unknown but may aggravate the decline of wild populations caused by the recently emerged natural recombinant myxoma virus.

microbiology↗