Biology subjects

Soares, R.

Publications and source records attributed to Soares, R..

11 recordsLinked to original sources

bioRxiv2026

Epithelial-Mesenchymal Transition Induces FAM134A-mediated ER-phagy to Regulate Procollagen Secretion

EMT is accompanied by extensive remodeling of the secretory pathway, yet how cancer cells maintain endoplasmic reticulum (ER) homeostasis during this transition remains unclear. Here we show that EMT activates ER-phagy as an adaptive mechanism to constrain secretory output. A genome-wide CRISPR activation screen unexpectedly identified EMT- and MET-associated transcription factors as opposing regulators of ER-phagy, and TGF{beta}-induced EMT robustly stimulated this pathway. Proteomic analysis revealed that ER-phagy regulates EMT-induced secretory protein degradation, including procollagens. Among FAM134 ER-phagy receptors, FAM134A uniquely mediated procollagen degradation through a non-canonical, LC3-independent mechanism. Loss of FAM134A diverted procollagen from degradation to secretion, resulting in a pro-invasive secretome, as conditioned media from FAM134A-deficient cells enhanced matrix invasion. These findings identify ER-phagy as a regulatory node that buffers EMT-associated secretory remodeling and reveal FAM134A as a context-specific suppressor of pro-invasive secretion.

cell biology↗

bioRxiv2025

The modular evolution of multiheme cytochromes c bucks the general trend observed in proteins

Multiheme cytochromes c are versatile enzymes which contribute to key steps in diverse biogeochemical cycles of elements and are also key players in microbial electrochemical technologies. We previously showed that these enzymes evolve by grafting and pruning of cytochrome c modules. Here, we extended our analysis and found that grafting can also involve the incorporation of domains of other protein families and of small peptide sequences that affect the heme coordination environment. We show that the addition of hemes to multiheme cytochromes c occurs exclusively by integrating heme-binding peptides and that gain of hemes is equally probable along the protein sequence. By contrast, heme loss occurs by the loss of heme-binding peptides and the accumulation of point mutations. Notably, heme-binding motif loss is disproportionately more prevalent at the position nearer to the N-terminus. This observation contrasts with the general trend observed in proteins, which are usually more conserved at the N-terminus, and likely reflects the way in which multiheme cytochromes c are assembled. Overall, this work completes the picture of how multiheme cytochromes c evolved to achieve the diversity of structures and functions found in nature, and sets them apart from other proteins with respect to the drivers for their evolution. This has contributed to the diversity of roles that they play in various biogeochemical cycles and has implications for engineering artificial variants to enhance biotechnological applications.

biochemistry↗

bioRxiv2024

Biomechanics of stem cell fate decisions in multilayered tissues

Tissue homeostasis relies on a precise balance of fate choices between renewal and differentiation, which is dysregulated during tumor initiation. Although much progress has been done over recent years to characterize the dynamics of cellular fate choices at the single cell level, their underlying mechanistic basis often remains unclear. In particular, although physical forces are increasingly characterized as regulators of cell behaviors, a unifying description of how global tissue mechanics interplays with local cellular fate choices is missing. Concentrating on skin epidermis as a paradigm for multilayered tissues with complex fate choices, we develop a 3D vertex-based model with proliferation restrained in the basal layer, showing that mechanics and competition for space naturally gives rise to homeostasis and neutral drift dynamics that are seen experimentally. We then explore the effect of introducing mechanical inhomogeneities, whereby subpopulations have differential tensions. We uncover that relatively small mechanical disparities can be sufficient to heavily tilt cellular towards symmetric renewal and exponential growth. Importantly, the simulations predict that such mechanical inhomogeneities are reflected by distinct morphological changes in single-cell shapes. This led us to derive a master relationship between two very different experimentally measurable parameters, cell shape and long-term clonal dynamics, which we validated using a model of basal cell carcinoma (BCC) consisting in clonal Smoothened overexpression in mouse tail epidermis. Altogether, we propose a theoretical framework to link mechanical forces, quantitative cellular morphologies and cellular fate outcomes in complex tissues.

biophysics↗

bioRxiv2024

A Survey of the Desulfuromonadia cytochromome provides a glimpse of the unexplored diversity of multiheme cytochromes in nature

Multiheme cytochromes (MHC) provide prokaryotes with a broad metabolic versatility that contributes to their role in the biogeochemical cycling of the elements. However, MHC were isolated and studied in detail only from a limited number of species. To obtain a broader view of the diversity of MHC, we employed bioinformatics tools to study the cytochromome encoded in the genomes of the Desulfuromonadia class. We found that MHC predicted to be extracellular are the least conserved and present higher diversity. Although the most prevalent MHC have homologues already characterized, nearly half of the MHC families in the Desulforomonadia class have no known homologues and AlphaFold2 was employed to predict their 3D structures. This work illuminates for the first time the universe of experimentally uncharacterized cytochromes that are likely to contribute to the metabolic versatility and to the fitness of Desulfuromonadia in diverse environmental conditions and to drive biotechnological applications.

biochemistry↗

bioRxiv2023

Unravelling a novel role for Cannabidivarin in the modulation of subventricular zone postnatal neurogenesis

Postnatal neurogenesis has been shown to rely on the endocannabinoid system. Here we aimed at unravelling the role of Cannabidivarin (CBDV), a non-psychoactive cannabinoid, with high affinity for the non-classical cannabinoid receptor TRPV1, on subventricular zone (SVZ) postnatal neurogenesis. Using the neurosphere assay, SVZ-derived neural stem/progenitor cells (NSPCs) were incubated with CBDV and/or 5-Iodoresinferotoxin (TRPV1 antagonist), and their role on cell viability, proliferation, and differentiation were dissected. CBDV was able to promote, through a TRPV1-dependent mechanism, cell survival, cell proliferation and neuronal differentiation. Furthermore, pulse-chase experiments revealed that CBDV-induced neuronal differentiation was a result of cell cycle exit of NSPCs. Regarding oligodendrocyte differentiation, CBDV inhibited oligodendrocyte differentiation and maturation. Since our data suggested that the CBDV-induced modulation of NSPCs acted via TRPV1, a sodium-calcium channel, and that intracellular calcium levels are known regulators of NSPCs fate and neuronal maturation, single cell calcium imaging was performed to evaluate the functional response of SVZ-derived cells. We observed that CBDV-responsive cells displayed a two-phase calcium influx profile, being the initial phase dependent on TRPV1 activation. Taken together, this work unveiled a novel and untapped neurogenic potential of CBDV via TRPV1 modulation. These findings pave the way to future neural stem cell biological studies and repair strategies by repurposing this non-psychoactive cannabinoid as a valuable therapeutic target.

neuroscience↗

bioRxiv2023

Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation

Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinsons disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1.

biochemistry↗

bioRxiv2022

A new paradigm of multiheme cytochrome evolution by grafting and pruning protein modules

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochromes c involved in metabolic pathways that contribute to the nitrogen, sulfur and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant ONR and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analised here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

biochemistry↗

bioRxiv2020

Thermogenic genes are blunted whereas brown adipose tissue identity is preserved in human obesity

Obesity associates with a reduction in cold-induced glucose tracer uptake in brown adipose tissue in humans, suggesting loss of thermogenic capacity. We therefore hypothesized that a whitening of BAT occurs in obesity and assessed the molecular characteristics of deep neck BAT in a cohort of 24 normal weight, 24 overweight and 22 obese individuals in comparison with subcutaneous abdominal white adipose tissue (WAT). We found that the major marker of BAT thermogenesis, UCP1, was associated with central but not general obesity. We performed transcriptomic analysis of BAT in a cohort of 27 individuals classified as normal weight, over-weight or obese, and additionally four subjects with type 2 diabetes (T2DM), dispersed among the 3 BMI groups. We identified 3204 differentially expressed genes between BAT and WAT in samples from normal weight individuals, including genes involved in thermogenesis, but also revealing differences in developmental and immune system related genes. In BAT from individuals with overweight or obesity, 202 genes were downregulated and 66 of these were involved in cellular respiratory pathways, likely reflecting previously observed reduction in thermogenic function with obesity. Importantly, most BAT selective genes were not affected, and isolated adipose progenitors differentiated into thermogenic adipocytes with equal frequency regardless of BMI group. In conclusion, our data suggest a retained BAT identity, with a selective reduction of thermogenic genes, in human obesity.

cell biology↗

bioRxiv2020

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress in Leishmania major

Lathosterol oxidase (LSO) catalyzes the formation of C5-C6 double bond in the synthesis of various types of sterols in mammals, fungi, plants and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C5-C6 desaturation, we generated a LSO-null mutant line (lso-) in Leishmania major, the causative agent for cutaneous leishmaniasis. Lso- parasites lacked the ergostane-based sterols commonly found in wild type L. major and instead accumulated equivalent sterol species without the C5-C6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso- mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alteration in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania. All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C5-C6 double bond is vital for the structure of sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress. IMPORTANCESterols are essential membrane components in eukaryotes and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. Lathosterol oxidase (aka sterol C5-desaturase) is required for the formation of C5-C6 double bond in the sterol core structure in mammals, fungi, protozoans, plants and algae. Functions of this C5-C6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major. Our data suggest that the C5-C6 double bond is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.

microbiology↗

bioRxiv2020

Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling

Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. Ser-Arg Repeat Protein Kinase (SRPK) is one such conserved eukaryotic kinase, which controls mRNA splicing. Surprisingly, we show that SRPK has acquired a novel function in regulating a neurodevelopmental ubiquitin signalling pathway. In mammalian embryonic stem cells, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of transcription factor substrates, thereby acting to restrain a neural gene expression programme that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signalling that ensures correct regulation of neurodevelopmental gene expression.

developmental biology↗

bioRxiv2018

A long-non-coding RNA, LINC00473, confers the human adipose tissue thermogenic phenotype through enhanced cAMP responsiveness.

Specialized adipocytes localized in distinct depots mediate the many physiological functions of adipose tissue. In humans, paucity of thermogenic adipocytes correlates with high metabolic disease risk, raising much interest in the mechanisms by which these cells arise. Here we report molecular signatures associated with adipocyte development in different human depots and identify a long non-coding RNA, LINC00473, as the transcript most closely associated with enrichment of thermogenic adipocytes. LINC00473 expression is low in subjects with obesity or type-2 diabetes and is highly correlated with cAMP signaling and mitochondrial oxidative phosphorylation pathways. LINC00473 is localized in the nucleus and the cytoplasm, and its knockdown impairs induction of UCP1 and mitochondrial respiration. These results reveal that depot-enriched genes that modulate responsiveness to external stimuli, specifically LINC00473, are important determinants of the adipose tissue thermogenic phenotype, and potential targets for metabolic disease therapy.

cell biology↗