Biology subjects

Costa-Filho, A. J.

Publications and source records attributed to Costa-Filho, A. J..

15 recordsLinked to original sources

bioRxiv2026

Exploring the effects of Golgi Reassembly and Stacking Proteins in lipid membranes

Golgi Reassembly and Stacking Proteins (GRASP) have been associated with Golgi-ribbon structure and unconventional vesicular protein secretion. In performing these functions, GRASPs consistently interact with membranes. The presence of lipid modifications, such as myristoylation, is a crucial consideration for obtaining detailed information about the interactions between GRASPs and membranes. Nonetheless, it has been overlooked in the literature so far. Here, we describe a reconstitution protocol for myristoylated human GRASP65 and GRASP55 in lipid model membranes, enabling investigation of their interactions using techniques ranging from structural characterization to spectroscopy and microscopy. Our results showed that myristoyl-anchored GRASPs can influence membrane dynamics, suggesting a possible role for their disordered SPR domain in this interaction.

biophysics↗

bioRxiv2025

Mapping high resolution, multidimensional phase diagrams of physiological protein condensates

Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of physiological protein condensates. We apply this approach to study two complex multicomponent condensate systems, stress granules and nucleoli, and dissect the physicochemical interactions that influence their stability. We further developed a machine learning pipeline to analyse condensate morphology which we use to reveal how mutations in the ALS-linked protein FUS remodels condensate phase landscapes. We identify liquid-to-solid transitions of mutant FUS within stress granules and nucleoli, and show that these transitions can be reversed by RNA aptamer-based interventions. Together, these findings establish ExVivo PhaseScan as a versatile tool for dissecting the physicochemical and pathological regulation of condensates, with potential to inform therapeutic strategies for diseases driven by aberrant phase transitions.

biophysics↗

bioRxiv2025

The role of N-terminal acetylation on biomolecular condensation

N-terminal acetylation (Nt-acetylation) is one of the most prevalent co-translational modifications in eukaryotes, affecting nearly 80% of the human proteome. Despite its ubiquity, the potential impact of this phenomenon on biomolecular condensation has been largely overlooked. Here, we uncover how this chemically subtle modification can exert broad and multifaceted control over phase behaviour, using Grh1, a Golgi-associated protein involved in stress-induced secretion in yeast, as a model system. We show that Nt-acetylation increases the saturation concentration for condensation, reduces droplet size and number, dampens pH sensitivity, weakens electrostatic contributions, and suppresses water dipolar relaxation within condensates, indicating reduced internal hydration and environmental responsiveness. These effects are accompanied by acetylation-dependent dimerisation and local structural changes, including a concentration-dependent gain in -helicity. Remarkably, co- condensation assays reveal that acetylated and non-acetylated forms of the same protein are only partially miscible, giving rise to core-shell architectures driven by differences in interfacial tension. Together, our findings highlight Nt-acetylation as a potent, generalizable regulator of condensate material properties, linking primary sequence chemistry to mesoscale organisation. Given its evolutionary conservation and prevalence across eukaryotic proteomes, Nt-acetylation may represent a widespread mechanism for modulating protein condensation in health and disease.

biophysics↗

bioRxiv2023

The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion

Eukaryotic cells have developed sophisticated mechanisms for biomolecule transport, particularly under stress conditions. This study focuses on the unconventional protein secretion (UPS) pathways activated during starvation stress, facilitating the export of soluble leaderless proteins. Specifically, we examine the role of the GRASP family, which is crucial in these processes but has largely unexplored mechanisms, especially regarding the biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results reveal that under the circumstances mimicking starvation conditions in yeast, liquid-liquid phase separation (LLPS) is instrumental in the self-association of Grh1, the yeast homologue of GRASP. This association is likely a precursor to the biogenesis of the so-called Compartment for Unconventional Protein Secretion. We demonstrate that Grh1s self-association involves complex interactions and is regulated by electrostatic, hydrophobic, and hydrogen-bonding forces. Importantly, our study shows that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations, a significant finding given the minimal interaction between these proteins under normal conditions. Additionally, we address how droplet ageing might affect the ability of cells to return to normal post-stress states. Our findings can potentially enhance our understanding of intracellular protein dynamics and their adaptive responses to stress in eukaryotic cells.

biophysics↗

bioRxiv2023

Biomolecular condensates of Chlorocatechol 1,2-Dioxygenase as enzymatic microreactors for the degradation of polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are molecules with two or more fused aromatic rings that occur naturally in the environment due to incomplete combustion of organic substances. However, the increased demand for fossil fuels in recent years has increased anthropogenic activity, contributing to the environmental concentration of PAHs. The enzyme chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is responsible for the breakdown of the aromatic ring of catechol, making it an interesting player in bioremediation strategies. Pp 1,2-CCD can tolerate a broader range of substrates, including halogenated compounds, than other dioxygenases. Here, we report the construction of a chimera protein able to form biomolecular condensates with potential application in bioremediation. The chimera protein was built by conjugating Pp 1,2-CCD to low complex domains (LCDs) derived from the DEAD-box protein Dhh1. We showed that the chimera could undergo liquid-liquid phase separation (LLPS), forming a protein-rich liquid droplet under different conditions (variable protein and PEG8000 concentrations and pH values), in which the protein maintained its structure and main biophysical properties. The condensates were active against 4-chlorocatechol, showing that the chimera droplets preserved the enzymatic activity of the native protein. The possible application of these microreactors in bioremediation is discussed.

biophysics↗

bioRxiv2021

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

The Golgi complex is a membranous organelle located in the heart of the eukaryotic secretory pathway. A subfamily of the Golgi matrix proteins, called GRASPs, are key players in the stress-induced unconventional secretion, the Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogs (GRASP55/GRASP65) and two coiled-coil Golgins (GM130/Golgin45), which interact with each other in vivo. Although essential for the Golgi ribbon formation and the increase in Golgi structural complexity, the molecular details leading to their appearance only in this subphylum are still unknown. Moreover, despite the new functionalities supported by the GRASP paralogy, little is known about the structural and evolutionary differences between these paralogues. In this context, we used ancestor sequence reconstruction and several biophysical/biochemical approaches to assess the evolution of the GRASP structure, flexibility, and how they started anchoring their Golgin partners. Our data showed that the Golgins appeared in evolution and were anchored by the single GRASP ancestor before gorasp gene duplication and divergence in Metazoans. After the gorasp divergence, variations inside the GRASP binding pocket determined which paralogue would recruit each Golgin partner (GRASP55 with Golgin45 and GRASP65 with GM130). These interactions are responsible for the proteins specific Golgi locations and the appearance of the Golgi ribbon. We also suggest that the capacity of GRASPs to form supramolecular structures is a long-standing feature, which likely affects GRASPs participation as a trigger of the stress-induced secretory pathway.

biophysics↗

bioRxiv2021

Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55

GRASP55 is a myristoylated protein localized in the medial/trans-Golgi faces and involved in the Golgi structure maintenance and the regulation of unconventional secretion pathways. It is believed that GRASP55 achieves its main functionalities in the Golgi organization by acting as a tethering factor and, when bound to the lipid bilayer, its orientation relative to the membrane surface is restricted to determine its proper trans-oligomerization. Despite the paramount role of myristoylation in GRASP function, the impact of such protein modification on the membrane-anchoring properties and the structural organization of GRASP remains elusive. Here, an optimized protocol for the myristoylation in E. coli of the membrane-anchoring domain of GRASP55 is presented. The biophysical properties of the myristoylated/non-myristoylated GRASP55 GRASP domain were characterized in a membrane-mimicking micellar environment. Although myristoylation did not cause any impact on the proteins secondary structure, according to our circular dichroism data, it had a significant impact on the proteins thermal stability and solubility. Electrophoresis of negatively charged liposomes incubated with the two GRASP55 constructions showed different electrophoretic mobility for the myristoylated anchored protein only, thus demonstrating that myristoylation is essential for the biological membrane anchoring. Molecular dynamics simulations were used to further explore the anchoring process in determining the restricted orientation of GRASPs in the membrane.

biophysics↗

bioRxiv2021

Structural and thermodynamic analyses of human TMED1 (p24γ1) Golgi dynamics

The transmembrane emp24 domain-containing proteins (TMED), also called p24 proteins, are members of a family of sorting receptors present in all representatives of the domain Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMEDs structure, oligomerization propensity, and biophysics of their interactions with the transport cargo. Here, we describe the first high-resolution structure of the Golgi dynamics (GOLD) domain of a TMED1 representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane are also presented.

biophysics↗

bioRxiv2021

In vivo amyloid-like fibrils produced under stress

The participation of amyloids in neurodegenerative diseases and functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. The autofluorescence from the extended {beta}-sheets of amyloids is here used to follow fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS), and here its participation also in heat shock response (HSR) is suggested. Fluorescence Lifetime Imaging (FLIM) is used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress is further supported by size exclusion chromatography and ultracentrifugation. The data show for the first time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.

biophysics↗

bioRxiv2019

Effects of nicotine on the thermodynamics of the DPPC phase coexistence region

Phase separation plays critical roles in several membrane functions, and reduction or disappearance of phase coexistence by action of membrane-interacting molecules have been implicated in membrane function impairment. Here, we applied differential scanning calorimetry, electron paramagnetic resonance (EPR), and non-linear least-squares (NLLS) spectral simulations to study the effects of nicotine, a parasympathomimetic drug, on the two-phase coexistence of dipalmitoyl phosphatidylcholine (DPPC) lipid membrane. The thermodynamic quantities describing the DPPC phase coexistence are temperature dependent, giving rise to non-linear vant Hoff behavior. Our results showed that nicotine preferentially binds to the fluid phase and modifies the enthalpy and entropy changes of the DPPC heat capacity profile, while marginally perturbing the homogeneous gel and fluid phases. An EPR/NLLS/vant Hoff analysis of the DPPC phase coexistence revealed that nicotine significantly modified the temperature dependence of the free energy change of the two-phase equilibrium from a cubic to a parabolic behavior, resulting in an alteration of the thermodynamical driving force and the balance of the non-covalent interactions of the lipids in equilibrium. The thermotropic behavior of the enthalpy, entropy, and heat capacity changes, as determined by EPR, indicated that nicotine modified the relative contributions of hydrogen-bonding, electrostatic interactions, and conformational entropy of the lipids to the thermodynamics of the phase coexistence. The predominantly entropically-driven gel-fluid transition in nicotine-free DPPC changes to a temperature-triggered entropically-driven or enthalpically-driven process in nicotine-bound DPPC. Further applications of this thermodynamic EPR/NLLS/vant Hoff analysis are discussed.

biophysics↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗

bioRxiv2019

Exploring Conformational Transitions and Free Energy Profiles of Proton Coupled Oligopeptide Transporters

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane helices that come together to form a \"V\" shaped transporter with a central substrate binding site. Proton coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF) and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding, however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands are still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both, proton and ligand, significantly change the conformational free energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations becames accessible. For the Glu300 Holo-PepTst, the presence of a second free energy minimum suggests that OF conformations are not only accessible, but also, stable. The differences in the free energy profiles demonstrate that transitions toward outward facing conformation occur only after protonation and, probably, this should be the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.\n\nGraphical TOC Entry\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/651646v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@8c8a84org.highwire.dtl.DTLVardef@da15bborg.highwire.dtl.DTLVardef@1c8dab0org.highwire.dtl.DTLVardef@18d87e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Exploring Structural Aspects of the Human Golgi Matrix Protein GRASP55 in Solution

In mammalian cells, the Golgi apparatus is a central hub for intracellular trafficking, sorting and post-translational modifications of proteins and lipids. The Golgi reassembly and stacking proteins (GRASPs) are somehow involved in the Golgi stacking, which is significant for the proper function of the Golgi apparatus, and also in unconventional protein secretion. However, the structural details on how GRASPs accomplish those tasks are still elusive. In this context, we have explored the biochemical and biophysical properties of the human full-length GRASP55 in solution. Sequence-based analyses and circular dichroism spectroscopy suggest that GRASP55 presents multiple intrinsically disordered sites, although keeping considerable contents of secondary structure. Size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS) studies show that GRASP55 are monomers in solution. Urea denaturation of GRASP55 suggests that the transition to the unfolded state is a cooperative process. Differential scanning calorimetry (DSC) analysis displays two endothermic transitions for GRASP55, indicating the existence of an intermediate state prior to unfolding. Thioflavin T fluorescence shows that GRASP55 can form protein aggregates/fibrils at the intermediate state. Transmission electron microscopy and fluorescence lifetime imaging microscopy prove that GRASP55 forms large amorphous aggregates but not amyloid-like fibrils in the intermediate state. The significance of these results could be helpful in discussing the proper function of human GRASP55 in the Golgi organization as well as unconventional secretion of proteins.

biophysics↗

bioRxiv2019

The GRASP domain in Golgi Reassembly and Stacking Proteins: differences and similarities between lower and higher Eukaryotes

The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flatted membrane sandwich kept together by a protein matrix. The correct mechanism controlling the Golgi cisternae assembly is not yet known, but it is already accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on both human GRASP55 and GRASP65 and compare them with GRASPs from lower eukaryotes (S. cerevisiae and C. neoformans). Our data suggest that both human GRASPs are essentially different from each other and GRASP65 is more similar to the subgroup of GRASPs from lower eukaryotes. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both funguses, while GRASP65 is located in the cis-Golgi. We suggest that the GRASP65 gene is more ancient and the paralogue GRASP55 might have appeared latter in evolution, together with the medial and trans Golgi faces in mammalians.

biophysics↗

bioRxiv2018

The yeast GRASP Grh1 displays a high polypeptide backbone mobility along with an amyloidogenic behavior

GRASPs are proteins involved in cell processes that seem paradoxical, such as being responsible for shaping the Golgi cisternae and also involved in unconventional secretion mechanisms that bypass the Golgi, among other functions in the cell. Despite its involvement in several relevant cell processes, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that is also observed in the GRASP from the yeast S. cerevisae (Grh1), which strongly suggests it may be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and amyloid fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.

biophysics↗