RESUMEN
The conformational variability of biological macromolecules can play an important role in their biological function. Therefore, understanding conformational variability is expected to be key for predicting the behavior of a particular molecule in the context of organism-wide studies. Several experimental methods have been developed and deployed for accessing this information, and computational methods are continuously updated for the profitable integration of different experimental sources. The outcome of this endeavor is conformational ensembles, which may vary significantly in properties and composition when different ensemble reconstruction methods are used, and this raises the issue of comparing the predicted ensembles against experimental data. In this article, we discuss a geometrical formulation to provide a framework for understanding the agreement of an ensemble prediction to the experimental observations.
Asunto(s)
Conformación Proteica , Proteínas , Proteínas/química , Modelos MolecularesRESUMEN
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/análisis , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Conformación Proteica , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Imagen por Resonancia MagnéticaRESUMEN
Intrinsically disordered proteins (IDPs) are significantly enriched in proline residues, which can populate specific local secondary structural elements called PPII helices, characterized by small packing densities. Proline is often thought to promote disorder, but it can participate in specific π·CH interactions with aromatic side chains resulting in reduced conformational flexibilities of the polypeptide. Differential local motional dynamics are relevant for the stabilization of preformed structural elements and can serve as nucleation sites for the establishment of long-range interactions. NMR experiments to probe the dynamics of proline ring systems would thus be highly desirable. Here we present a pulse scheme based on 13C detection to quantify dipole-dipole cross-correlated relaxation (CCR) rates at methylene CH2 groups in proline residues. Applying 13C-CON detection strategy provides exquisite spectral resolution allowing applications also to high molecular weight IDPs even in conditions approaching the physiological ones. The pulse scheme is illustrated with an application to the 220 amino acids long protein Osteopontin, an extracellular cytokine involved in inflammation and cancer progression, and a construct in which three proline-aromatic sequence patches have been mutated.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Humanos , Imagen por Resonancia Magnética , Frecuencia Cardíaca , Inflamación , Conformación MolecularRESUMEN
Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5ß interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5ß and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5ß(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5ß protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5ß(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5ß interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5ß. Expression of LL5ß(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.
Asunto(s)
Membrana Celular , Movimiento Celular , Inmunoprecipitación , Células MDA-MB-231 , HumanosRESUMEN
An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.
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Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Conformación ProteicaRESUMEN
The knowledge of interactions between different molecules is undoubtedly the driving force of all contemporary biomedical and biological sciences. Chemical biology/biological chemistry has become an important multidisciplinary bridge connecting the perspectives of chemistry and biology to the study of small molecules/peptidomimetics and their interactions in biological systems. Advances in structural biology research, in particular linking atomic structure to molecular properties and cellular context, are essential for the sophisticated design of new medicines that exhibit a high degree of druggability and very importantly, druglikeness. The authors of this contribution are outstanding scientists in the field who provided a brief overview of their work, which is arranged from in silico investigation through the characterization of interactions of compounds with biomolecules to bioactive materials.
Asunto(s)
Biología MolecularRESUMEN
Novel and efficient strategies need to be developed to interfere with the SARS-CoV-2 virus. One of the most promising pharmaceutical targets is the nucleocapsid protein (N), responsible for genomic RNA packaging. N is composed of two folded domains and three intrinsically disordered regions (IDRs). The globular RNA binding domain (NTD) and the tethered IDRs are rich in positively charged residues. The study of the interaction of N with polyanions can thus help to elucidate one of the key driving forces responsible for its function, i.e., electrostatics. Heparin, one of the most negatively charged natural polyanions, has been used to contrast serious cases of COVID-19 infection, and we decided to study its interaction with N at the molecular level. We focused on the NTR construct, which comprises the NTD and two flanking IDRs, and on the NTD construct in isolation. We characterized this interaction using different nuclear magnetic resonance approaches and isothermal titration calorimetry. With these tools, we were able to identify an extended surface of NTD involved in the interaction. Moreover, we assessed the importance of the IDRs in increasing the affinity for heparin, highlighting how different tracts of these flexible regions modulate the interaction.
Asunto(s)
Enoxaparina , Proteínas de la Nucleocápside , SARS-CoV-2 , COVID-19 , Enoxaparina/farmacología , Humanos , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Polielectrolitos , ARN , SARS-CoV-2/efectos de los fármacosRESUMEN
The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1-248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , ARN Viral/metabolismoRESUMEN
Thanks to recent improvements in NMR spectrometer hardware and pulse sequence design, modern 13C NMR has become a useful tool for biomolecular applications. The complete assignment of a protein can be accomplished by using 13C detected multinuclear experiments and it can provide unique information relevant for the study of a variety of different biomolecules including paramagnetic proteins and intrinsically disordered proteins. A wide range of NMR observables can be measured, concurring to the structural and dynamic characterization of a protein in isolation, as part of a larger complex, or even inside a living cell. We present the different properties of 13C with respect to 1H, which provide the rationale for the experiments developed and their application, the technical aspects that need to be faced, and the many experimental variants designed to address different cases. Application areas where these experiments successfully complement proton NMR are also described.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Imagen por Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular/métodos , ProtonesRESUMEN
Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a ß-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200-310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.
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Amiloide/metabolismo , Virus Hendra/metabolismo , Transición de Fase , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Rojo Congo/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Hidrogeles/química , Espectroscopía de Resonancia Magnética , Dominios Proteicos , Dispersión del Ángulo Pequeño , Proteínas Virales/ultraestructura , Difracción de Rayos XRESUMEN
The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.
RESUMEN
The nucleocapsid protein N from SARS-CoV-2 is one of the most highly expressed proteins by the virus and plays a number of important roles in the transcription and assembly of the virion within the infected host cell. It is expected to be characterized by a highly dynamic and heterogeneous structure as can be inferred by bioinformatics analyses as well as from the data available for the homologous protein from SARS-CoV. The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus/química , Espectroscopía de Resonancia Magnética , SARS-CoV-2/química , Isótopos de Carbono , Biología Computacional , Hidrógeno , Isótopos de Nitrógeno , Fosfoproteínas/química , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
NMR represents a key spectroscopic technique that contributes to the emerging field of highly flexible, intrinsically disordered proteins (IDPs) or protein regions (IDRs) that lack a stable three-dimensional structure. A set of exclusively heteronuclear NMR experiments tailored for proline residues, highly abundant in IDPs/IDRs, are presented here. They provide a valuable complement to the widely used approach based on amide proton detection, filling the gap introduced by the lack of amide protons in proline residues within polypeptide chains. The novel experiments have very interesting properties for the investigations of IDPs/IDRs of increasing complexity.
RESUMEN
Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.
Asunto(s)
Infecciones por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteína de Unión a CREB/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Adenoviridae/genética , Infecciones por Adenoviridae/genética , Infecciones por Adenoviridae/virología , Proteínas E1A de Adenovirus/genética , Proteína de Unión a CREB/genética , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Unión Proteica , Dominios ProteicosRESUMEN
Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-ß (Aß) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aß and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aß while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aß-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Descubrimiento de Drogas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos de Péptidos/metabolismoRESUMEN
Copper/zinc superoxide dismutase (SOD) is a homodimeric metalloenzyme that has been extensively studied as a benchmark for structure-function relationships in proteins, in particular because of its implication in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis. Here, we investigate microcrystalline preparations of two differently metalated forms of SOD, namely, the fully mature functional Cu,Zn state and the E,Zn-SOD state in which the Cu site is empty. By using solid-state NMR with fast magic-angle spinning (MAS) at high magnetic fields (1H Larmor frequency of 800-1000 MHz), we quantify motions spanning a dynamic range from ns to ms. We determine that metal ion uptake does not act as a rigidification element but as a switch redistributing motional processes on different time scales, with coupling of the dynamics of histidine side chains and those of remote key backbone elements of the protein.
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Esclerosis Amiotrófica Lateral/metabolismo , Cobre/química , Histidina/química , Superóxido Dismutasa/química , Zinc/química , Sitios de Unión , Cristalización , Humanos , Cinética , Campos Magnéticos , Espectroscopía de Resonancia Magnética , Metaloproteínas/química , Modelos Moleculares , Conformación Proteica , Multimerización de ProteínaRESUMEN
Using SAXS and NMR spectroscopy, we herein provide a high-resolution description of the intrinsically disordered N-terminal domain (PNT, aa 1-406) shared by the Nipah virus (NiV) phosphoprotein (P) and V protein, two key players in viral genome replication and in evasion of the host innate immune response, respectively. The use of multidimensional NMR spectroscopy allowed us to assign as much as 91% of the residues of this intrinsically disordered domain whose size constitutes a technical challenge for NMR studies. Chemical shifts and nuclear relaxation measurements provide the picture of a highly flexible protein. The combination of SAXS and NMR information enabled the description of the conformational ensemble of the protein in solution. The present results, beyond providing an overall description of the conformational behavior of this intrinsically disordered region, also constitute an asset for obtaining atomistic information in future interaction studies with viral and/or cellular partners. The present study can thus be regarded as the starting point towards the design of inhibitors that by targeting crucial protein-protein interactions involving PNT might be instrumental to combat this deadly virus.
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Fosfoproteínas/química , Proteínas Virales/química , Proteínas Estructurales Virales/química , Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/metabolismo , Conformación Proteica , Dominios Proteicos , Dispersión del Ángulo Pequeño , Proteínas Virales/metabolismo , Proteínas Estructurales Virales/metabolismo , Difracción de Rayos XRESUMEN
Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10-4-10-5 Å. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10-1 Å. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign 1H, 13C, and 15N resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the CoII coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the CoII/ZnII centers in these proteins, which allows them to play a purely structural role.
Asunto(s)
Cobalto/química , Complejos de Coordinación/química , Metaloproteínas/química , Superóxido Dismutasa-1/química , Zinc/química , Sitios de Unión , Humanos , Resonancia Magnética Nuclear BiomolecularRESUMEN
Many properties of intrinsically disordered proteins (IDPs), or protein regions (IDRs), are modulated by the nature of amino acid side chains as well as by local solvent exposure. We propose a set of exclusively heteronuclear NMR experiments to investigate these features in different experimental conditions that are relevant for physiological function. The proposed approach is generally applicable to many IDPs/IDRs whose assignment is available in the Biological Magnetic Resonance Bank (BMRB) to investigate how their properties are modulated by different, physiologically relevant conditions. The experiments, tested on α-synuclein, are then used to investigate how α-synuclein senses Ca2+ concentration jumps associated with the transmission of nerve signals. Novel modules in the primary sequence of α-synuclein optimized for calcium sensing in highly flexible, disordered protein segments are identified.
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Calcio/química , Resonancia Magnética Nuclear Biomolecular , alfa-Sinucleína/química , Secuencias de Aminoácidos , Calcio/metabolismo , Isótopos de Carbono/química , Concentración de Iones de Hidrógeno , Iones/química , Temperatura , Agua/química , alfa-Sinucleína/metabolismoRESUMEN
Crosstalk between cellular pathways is often mediated through scaffold proteins that function as platforms for the assembly of signaling complexes. Based on yeast two-hybrid analysis, we report here the interaction between two complex scaffold proteins, CREB-binding protein (CBP) and the Ras GTPase-activating-like protein 1 (IQGAP1). Dissection of the interaction between the two proteins reveals that the central, thus far uncharacterized, region of IQGAP1 interacts with the HAT domain and the C-terminal intrinsically disordered region of CBP (termed ID5). Structural analysis of ID5 by solution NMR spectroscopy and SAXS reveals the presence of two regions with pronounced helical propensity. The ID5 region(s) involved in the interaction of nanomolar affinity were delineated by solution NMR titrations and pull-down assays. Moreover, we found that IQGAP1 acts as an inhibitor of the histone acetyltransferase (HAT) activity of CBP. In in vitro assays, the CBP-binding region of IQGAP1 positively and negatively regulates the function of HAT proteins of different families including CBP, KAT5 and PCAF. As many signaling pathways converge on CBP and IQGAP1, their interaction provides an interface between transcription regulation and the coordination of cytoskeleton. Disruption or alteration of the interaction between these scaffold proteins may lead to cancer development or metastatic processes, highlighting the importance of this interaction.