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1.
Br J Pharmacol ; 2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39209310

RESUMO

G protein-coupled receptors (GPCRs) play a crucial role in cell function by transducing signals from the extracellular environment to the inside of the cell. They mediate the effects of various stimuli, including hormones, neurotransmitters, ions, photons, food tastants and odorants, and are renowned drug targets. Advancements in structural biology techniques, including X-ray crystallography and cryo-electron microscopy (cryo-EM), have driven the elucidation of an increasing number of GPCR structures. These structures reveal novel features that shed light on receptor activation, dimerization and oligomerization, dichotomy between orthosteric and allosteric modulation, and the intricate interactions underlying signal transduction, providing insights into diverse ligand-binding modes and signalling pathways. However, a substantial portion of the GPCR repertoire and their activation states remain structurally unexplored. Future efforts should prioritize capturing the full structural diversity of GPCRs across multiple dimensions. To do so, the integration of structural biology with biophysical and computational techniques will be essential. We describe in this review the progress of nuclear magnetic resonance (NMR) to examine GPCR plasticity and conformational dynamics, of atomic force microscopy (AFM) to explore the spatial-temporal dynamics and kinetic aspects of GPCRs, and the recent breakthroughs in artificial intelligence for protein structure prediction to characterize the structures of the entire GPCRome. In summary, the journey through GPCR structural biology provided in this review illustrates how far we have come in decoding these essential proteins architecture and function. Looking ahead, integrating cutting-edge biophysics and computational tools offers a path to navigating the GPCR structural landscape, ultimately advancing GPCR-based applications.

2.
J Agric Food Chem ; 72(26): 14521-14529, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38906535

RESUMO

Chemosensory membrane proteins such as G-protein-coupled receptors (GPCRs) drive flavor perception of food formulations. To achieve this, a detailed understanding of the structure and function of these membrane proteins is needed, which is often limited by the extraction and purification methods involved. The proposed nanodisc methodology helps overcome some of these existing challenges such as protein stability and solubilization along with their reconstitution from a native cell-membrane environment. Being well-established in structural biology procedures, nanodiscs offer this elegant solution by using, e.g., a membrane scaffold protein (MSP) or styrene-maleic acid (SMA) polymer, which interacts directly with the cell membrane during protein reconstitution. Such derived proteins retain their biophysical properties without compromising the membrane architecture. Here, we seek to show that these lipidic systems can be explored for insights with a focus on chemosensory membrane protein morphology and structure, conformational dynamics of protein-ligand interactions, and binding kinetics to answer pending questions in flavor research. Additionally, the compatibility of nanodiscs across varied (labeled or label-free) techniques offers significant leverage, which has been highlighted here.


Assuntos
Proteínas de Membrana , Proteínas de Membrana/química , Nanoestruturas/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Aromatizantes/química , Humanos
3.
Nat Food ; 5(4): 281-287, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38605131

RESUMO

Food texture, along with taste and odour, is an important factor in determining food flavour. However, the physiological properties of oral texture perception require greater examination and definition. Here we explore recent trends and perspectives related to mouthfeel and its relevance in food flavour perception, with an emphasis on the biophysical point of view and methods. We propose that atomic force microscopy, combined with other biophysical techniques and more traditional food science approaches, offers a unique opportunity to study the mechanisms of mouthfeel at cellular and molecular levels. With this knowledge, food composition could be modified to develop healthier products by limiting salt, sugar, fat and calories while maintaining sensory qualities and consumer acceptance.


Assuntos
Microscopia de Força Atômica , Boca , Percepção Gustatória , Humanos , Paladar/fisiologia , Percepção Gustatória/fisiologia
4.
ACS Nanosci Au ; 4(2): 136-145, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38644967

RESUMO

The SARS-CoV-2 pandemic spurred numerous research endeavors to comprehend the virus and mitigate its global severity. Understanding the binding interface between the virus and human receptors is pivotal to these efforts and paramount to curbing infection and transmission. Here we employ atomic force microscopy and steered molecular dynamics simulation to explore SARS-CoV-2 receptor binding domain (RBD) variants and angiotensin-converting enzyme 2 (ACE2), examining the impact of mutations at key residues upon binding affinity. Our results show that the Omicron and Delta variants possess strengthened binding affinity in comparison to the Mu variant. Further, using sera from individuals either vaccinated or with acquired immunity following Delta strain infection, we assess the impact of immunity upon variant RBD/ACE2 complex formation. Single-molecule force spectroscopy analysis suggests that vaccination before infection may provide stronger protection across variants. These results underscore the need to monitor antigenic changes in order to continue developing innovative and effective SARS-CoV-2 abrogation strategies.

5.
Proc Natl Acad Sci U S A ; 120(24): e2219404120, 2023 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-37276413

RESUMO

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.


Assuntos
Orthoreovirus , Reoviridae , Animais , Humanos , Receptor Nogo 1/metabolismo , Ligação Viral , Proteínas Virais/metabolismo , Ligantes , Reoviridae/metabolismo , Orthoreovirus/metabolismo , Receptores Virais/metabolismo , Mamíferos/metabolismo
6.
Proc Natl Acad Sci U S A ; 120(21): e2220741120, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37186838

RESUMO

Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.


Assuntos
Orthoreovirus , Reoviridae , Animais , Proteínas do Capsídeo/química , Reoviridae/metabolismo , Orthoreovirus/metabolismo , Proteínas Virais/metabolismo , Ligação Viral , Anticorpos Antivirais , Mamíferos/metabolismo
7.
Nat Commun ; 14(1): 2615, 2023 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-37147336

RESUMO

Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain.


Assuntos
Orthoreovirus de Mamíferos , Receptores Imunológicos , Receptores Virais , Infecções por Reoviridae , Animais , Humanos , Camundongos , Anticorpos Antivirais , Orthoreovirus de Mamíferos/fisiologia , Receptores Imunológicos/metabolismo , Infecções por Reoviridae/metabolismo , Receptores Virais/metabolismo
8.
J Struct Biol ; 215(2): 107963, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37044358

RESUMO

The application of atomic force microscopy (AFM) for functional imaging and manipulating biomolecules at all levels of organization has enabled great progress in the structural biology field over the last decades, contributing to the discovery of novel structural entities of biological significance across many disciplines ranging from biochemistry, biomedicine and biophysics to molecular and cell biology, up to food systems and beyond. AFM has the capability to generate high-resolution topographic images spanning from the submolecular to the (sub)cellular range and can probe biochemical and biophysical sample properties in close to native conditions with excellent temporal resolution. Instrumental developments in the past decade enable dynamical structural and conformational studies of single biomolecules and new techniques for structural and chemical modification of the AFM probe have converted the cantilever into a versatile tool to study different biological phenomena, such as the mechanical stability of biomolecular complexes or the force induced dynamic changes of mechanically stressed proteins at the nanoscopic level. To improve the functionality of AFM and approach dynamic processes of complex biological systems ex vivo, AFM is combined with complementary microscopy, nanoscopy and spectroscopy tools. These multimethodological approaches provide unprecedented possibilities of probing physical, chemical and biological properties of complex cellular systems with high spatio-temporal resolution, leading to novel applications that correlate structural results with functional biochemical, biophysical, immunological, or genetic data of the system under study.


Assuntos
Biologia , Fenômenos Mecânicos , Microscopia de Força Atômica/métodos
9.
Nano Lett ; 23(4): 1496-1504, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36758952

RESUMO

Despite intense scrutiny throughout the pandemic, development of efficacious drugs against SARS-CoV-2 spread remains hindered. Understanding the underlying mechanisms of viral infection is fundamental for developing novel treatments. While angiotensin converting enzyme 2 (ACE2) is accepted as the key entry receptor of the virus, other infection mechanisms exist. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and its counterpart DC-SIGN-related (DC-SIGNR, also known as L-SIGN) have been recognized as possessing functional roles in COVID-19 disease and binding to SARS-CoV-2 has been demonstrated previously with ensemble and qualitative techniques. Here we examine the thermodynamic and kinetic parameters of the ligand-receptor interaction between these C-type lectins and the SARS-CoV-2 S1 protein using force-distance curve-based AFM and biolayer interferometry. We evidence that the S1 receptor binding domain is likely involved in this bond formation. Further, we employed deglycosidases and examined a nonglycosylated S1 variant to confirm the significance of glycosylation in this interaction. We demonstrate that the high affinity interactions observed occur through a mechanism distinct from that of ACE2.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Ligação Proteica
11.
Nat Commun ; 13(1): 2564, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35538121

RESUMO

The recent emergence of highly transmissible SARS-CoV-2 variants illustrates the urgent need to better understand the molecular details of the virus binding to its host cell and to develop anti-viral strategies. While many studies focused on the role of the angiotensin-converting enzyme 2 receptor in the infection, others suggest the important role of cell attachment factors such as glycans. Here, we use atomic force microscopy to study these early binding events with the focus on the role of sialic acids (SA). We show that SARS-CoV-2 binds specifically to 9-O-acetylated-SA with a moderate affinity, supporting its role as an attachment factor during virus landing to cell host surfaces. For therapeutic purposes and based on this finding, we have designed novel blocking molecules with various topologies and carrying a controlled number of SA residues, enhancing affinity through a multivalent effect. Inhibition assays show that the AcSA-derived glycoclusters are potent inhibitors of cell binding and infectivity, offering new perspectives in the treatment of SARS-CoV-2 infection.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Sítios de Ligação , Humanos , Ácido N-Acetilneuramínico , Ligação Proteica , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
12.
Chem Commun (Camb) ; 58(33): 5072-5087, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35315846

RESUMO

Understanding biological interactions at a molecular level grants valuable information relevant to improving medical treatments and outcomes. Among the suite of technologies available, Atomic Force Microscopy (AFM) is unique in its ability to quantitatively probe forces and receptor-ligand interactions in real-time. The ability to assess the formation of supramolecular bonds and intermediates in real-time on surfaces and living cells generates important information relevant to understanding biological phenomena. Combining AFM with fluorescence-based techniques allows for an unprecedented level of insight not only concerning the formation and rupture of bonds, but understanding medically relevant interactions at a molecular level. As the ability of AFM to probe cells and more complex models improves, being able to assess binding kinetics, chemical topographies, and garner spectroscopic information will likely become key to developing further improvements in fields such as cancer, nanomaterials, and virology. The rapid response to the COVID-19 crisis, producing information regarding not just receptor affinities, but also strain-dependent efficacy of neutralizing nanobodies, demonstrates just how viable and integral to the pre-clinical development of information AFM techniques are in this era of medicine.


Assuntos
COVID-19 , Nanoestruturas , Humanos , Cinética , Ligantes , Microscopia de Força Atômica/métodos
13.
Nano Lett ; 22(4): 1641-1648, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35108019

RESUMO

Ebola virus (EBOV) is responsible for several outbreaks of hemorrhagic fever with high mortality, raising great public concern. Several cell surface receptors have been identified to mediate EBOV binding and internalization, including phosphatidylserine (PS) receptors (TIM-1) and C-type lectin receptors (DC-SIGNR). However, the role of TIM-1 during early cell surface binding remains elusive and in particular whether TIM-1 acts as a specific receptor for EBOV. Here, we used force-distance curve-based atomic force microscopy (FD-based AFM) to quantify the binding between TIM-1/DC-SIGNR and EBOV glycoprotein (GP) and observed that both receptors specifically bind to GP with high-affinity. Since TIM-1 can also directly interact with PS at the single-molecule level, we also confirmed that TIM-1 acts as dual-function receptors of EBOV. These results highlight the direct involvement of multiple high-affinity receptors in the first steps of binding to cell surfaces, thus offering new perspectives for the development of anti-EBOV therapeutic molecules.


Assuntos
Ebolavirus , Ebolavirus/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Ligação Viral
14.
ACS Nano ; 16(1): 306-316, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34957816

RESUMO

Silica nanoparticles (SiNP) trigger a range of innate immune responses in relevant essential organs, such as the liver and the lungs. Inflammatory reactions, including NLRP3 inflammasome activation, have been linked to particulate materials; however, the molecular mechanisms and key actors remain elusive. Although many receptors, including several scavenger receptors, were suggested to participate in SiNP cellular uptake, mechanistic evidence of their role on innate immunity is lacking. Here we present an atomic force microscopy-based approach to physico-mechanically map the specific interaction occurring between nanoparticles and scavenger receptor A1 (SRA1) in vitro on living lung epithelial cells. We find that SiNP recognition by SRA1 on human macrophages plays a key role in mediating NLRP3 inflammasome activation, and we identify cellular mechanical changes as clear indicators of inflammasome activation in human macrophages, greatly advancing our knowledge on the interplay among nanomaterials and innate immunity.


Assuntos
Inflamassomos , Nanopartículas , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Macrófagos/metabolismo , Imunidade Inata , Dióxido de Silício/metabolismo
15.
Nat Commun ; 12(1): 6977, 2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34848718

RESUMO

Despite an unprecedented global gain in knowledge since the emergence of SARS-CoV-2, almost all mechanistic knowledge related to the molecular and cellular details of viral replication, pathology and virulence has been generated using early prototypic isolates of SARS-CoV-2. Here, using atomic force microscopy and molecular dynamics, we investigated how these mutations quantitatively affected the kinetic, thermodynamic and structural properties of RBD-ACE2 complex formation. We observed for several variants of concern a significant increase in the RBD-ACE2 complex stability. While the N501Y and E484Q mutations are particularly important for the greater stability, the N501Y mutation is unlikely to significantly affect antibody neutralization. This work provides unprecedented atomistic detail on the binding of SARS-CoV-2 variants and provides insight into the impact of viral mutations on infection-induced immunity.


Assuntos
Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/farmacologia , COVID-19/terapia , COVID-19/virologia , Humanos , Cinética , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Termodinâmica
16.
Nano Lett ; 21(22): 9720-9728, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34762801

RESUMO

Breast cancer is the most common cancer in women. Although current therapies have increased survival rates for some breast cancer types, other aggressive invasive breast cancers remain difficult to treat. As the onset of breast cancer is often associated with the appearance of extracellular markers, these could be used to better target therapeutic agents. Here, we demonstrated by nanobiophysical approaches that overexpression of α-sialylated glycans in breast cancer provides an opportunity to combat cancer cells with oncolytic reoviruses. Notably, a correlation between cellular glycan expression and the mechanical properties of reovirus attachment and infection is observed in a serotype-dependent manner. Furthermore, we enhance the infectivity of reoviruses in malignant cells by the coinjection of α-sialylated glycans. In conclusion, this study supports both the use of reoviruses as an oncolytic agent in nanomedicine and the role of α-sialylated glycans as adjuvants in oncolysis, offering new perspective in oncolytic cancer therapy.


Assuntos
Neoplasias da Mama , Reoviridae , Neoplasias da Mama/terapia , Feminino , Humanos , Polissacarídeos
17.
Nano Lett ; 21(12): 4950-4958, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34125553

RESUMO

PIEZO1 ion channels are activated by mechanical stimuli, triggering intracellular chemical signals. Recent structural studies suggest that plasma membrane tension or local curvature changes modulate PIEZO1 channel gating and activation. However, whether PIEZO1 localization is governed by tension gradients or long-range mechanical perturbations across the cells is still unclear. Here, we probe the nanoscale localization of PIEZO1 on red blood cells (RBCs) at high resolution (∼30 nm), and we report for the first time the existence of submicrometric PIEZO1 clusters in native conditions. Upon interaction with Yoda1, an allosteric modulator, PIEZO1 clusters increase in abundance in regions of higher membrane tension and lower curvature. We further show that PIEZO1 ion channels interact with the spectrin cytoskeleton in both resting and activated states. Our results point toward a strong interplay between plasma membrane tension gradients, curvature, and cytoskeleton association of PIEZO1.


Assuntos
Canais Iônicos , Fenômenos Mecânicos , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Microscopia Confocal
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