Biology subjects

Esposito, D.

Publications and source records attributed to Esposito, D..

At least 19 recordsLinked to original sources

bioRxiv2025

An In Vitro BRAF Activation Assay Elucidates Molecular Mechanisms Driving the Disassembly of the Autoinhibited BRAF State

The RAF kinases (ARAF, BRAF and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-32:MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential. Significance StatementBRAF is a central intermediate in RAS pathway signaling, and its activity is often elevated in human cancers and RASopathy disorders. Due to the complexity of BRAF activation, identifying compounds that sustainably inhibit BRAF function has proven difficult, emphasizing the need for a more comprehensive understanding of BRAF regulation. Here, we have developed an in vitro BRAF activation assay that elucidates key steps in this process. Our findings demonstrate that RAS binding not only recruits BRAF to the plasma membrane but initiates the disassembly of the autoinhibited monomer, which in the context of the membrane, facilitates BRAF dimerization and activation. This assay advances our understanding of BRAF regulation and provides a novel platform for drug discovery efforts targeting BRAF.

biochemistry↗

bioRxiv2025

Conformational dynamics of the bacterial E3 ligase SspH1

The SspH/IpaH family of novel E3 ligases (NELs) are found in a number of Gram-negative bacteria and are used to target host enzymes for degradation to support pathogenesis. These E3 enzymes are autoinhibited in the absence of substrate and different models for release of autoinhibition have been suggested. However, many of the molecular details of individual steps during the ubiquitin transfer reaction remain unknown. Here, we present the crystal structure of Salmonella SspH1 and an analysis of the solution properties of SspH1 on its own and in complex with substrate and ubiquitin. Our data show that SspH1 exists in a conformational equilibrium between open and closed states and that substrate binding only modulates the distribution of these states but does not induce major conformational changes. This suggests that additional mechanisms must exist to bring the substrates close to the active site to mediate transfer of ubiquitin from the E3[~]Ub conjugate.

biochemistry↗

bioRxiv2025

Hyper-responsiveness of cancer stem cells to microenvironmental cues controls metastasis and therapy response through YAP/TAZ/TEAD

Cancer stem cells (CSCs) are key drivers of metastasis and therapy resistance but have been challenging to visualize and study in situ. Using a fluorescent CSC reporter, we observed very different population dynamics for CSCs and nonCSCs during metastatic lung colonization in breast cancer models. CSC expansive self-renewal drives early lesion formation before switching to a maintenance mode of balanced self-renewal and differentiation, whereupon nonCSC proliferation takes over as the main driver of metastatic expansion. Mechanistic analyses showed that CSCs are hyper-responsive to microenvironmental cues such as cell crowding and nutrient availability, suggesting a novel role for CSCs as sensors and early responders to fluctuating local conditions in the tumor. Incoming signals converge on YAP/TAZ/TEAD, with heightened CSC sensitivity and response supported by elevated receptor expression and increased chromatin accessibility around enhancers with TEAD binding sites. Targeting inputs to the YAP/TAZ/TEAD node reversed chemotherapy-induced enrichment of CSCs in lung metastases. HighlightsO_LIDifferent population dynamics for breast cancer stem cells (CSCs) and their differentiated progeny in early metastatic colonization C_LIO_LICSCs are hyper-responsive to microenvironmental cues and serve as sensors of local conditions for the tumor C_LIO_LIMany microenvironmental inputs converge on YAP/TAZ to regulate self-renewal vs differentiation fate decisions in the CSC C_LIO_LITargeting YAP/TAZ input pathways blocks chemotherapy-induced enrichment of CSCs C_LI

cancer biology↗

bioRxiv2025

Salmonella effector kinase SteC is activated by host-mediated phosphorylation

The pathogen Salmonella, which causes significant human morbidity and mortality, encodes an effector kinase, SteC, which mediates actin polymerisation and cell migration. Given the minimal nature of its kinase domain, it remains unclear how SteC is catalytically active and how this activity is regulated. Here, we show that SteC is activated following the phosphorylation of the highly conserved S379 residue by a host kinase. Phosphorylation of S379 dramatically increases nucleotide binding affinity of SteC, enabling substrate phosphorylation and promoting actin polymerisation. Further mutational analysis identified the functional role of HD and DGD motifs that likely mimic the HxD and DFG motifs of eukaryotic kinases. Meanwhile, the C-tail of SteC, encompassing amino acids 429-457, is essential for function following translocation from Salmonella, but dispensable for catalysis in vitro. Overall, our findings uncover two previously unappreciated mechanisms that mediate the activity of the only Salmonella effector kinase within the host. HIGHLIGHTSSteC is phosphorylated at S379 in mammalian cells SteC phosphorylation is critical for kinase activity and actin polymerisation Phosphorylation at S379 promotes nucleotide binding The C-tail of SteC is not required for catalysis but is essential for activity in infected cells

microbiology↗

bioRxiv2024

Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRASG12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRASG12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

cancer biology↗

bioRxiv2024

Identification of RING E3 pseudoligases in the TRIM protein family

TRIpartite Motif (TRIM) family proteins have diverse roles across a broad variety of cellular functions, which are largely presumed to depend on their ubiquitin E3 ligase activity, conferred by a RING domain. However, recent reports have shown that some TRIMs lack detectable ubiquitination activity in isolation, despite containing a RING domain. Here, we present parallel in cellulo, in vitro, and in silico structure-function analyses of the ubiquitin E3 ligase activity and RING domain structural characteristics of whole TRIM protein family. In-depth follow-up studies of this comprehensive dataset reveals a number of pseudoligases, whose RING domains have structurally diverged at either the homodimerisation or E2[~]ubiquitin interfaces, thereby disrupting their ability to catalyse ubiquitin transfer. Together, these data raise intriguing open questions regarding the unknown TRIM functions in physiology and disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/614675v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@7c851org.highwire.dtl.DTLVardef@f06afdorg.highwire.dtl.DTLVardef@3cd7org.highwire.dtl.DTLVardef@19a60e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38{+/-}10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

biophysics↗

bioRxiv2023

The Multidrug Resistance Transporter P-glycoprotein Confers Resistance to Ferroptosis Inducers

Ferroptosis is a form of cell death caused by direct or indirect inhibition of glutathione peroxidase 4 that leads to lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding resistance mechanisms, particularly their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. Given the role that ABC transporters play in absorption, distribution, and excretion of many drugs, characterizing these interactions could provide information regarding oral bioavailability and brain penetration and may predict drug-drug interactions. Using ferroptosis-sensitive A673 cells transfected to express P-gp or ABCG2, we found that P-gp overexpression was able to confer resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. Results were confirmed with OVCAR8-derived NCI/ADR-RES cells that overexpress P-gp, where the P-gp inhibitor valspodar completely inhibited resistance to the FINs. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. At a concentration of 10 M, the FINs ML-162, GPX inhibitor 26a, and PACMA31 were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells and GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. Expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions. SIGNIFICANCE STATEMENTWhile several small-molecule ferroptosis inducers have been described, little work has addressed potential interactions with ABC transporters such as P-glycoprotein or ABCG2 that might limit bioavailability or brain penetration. We find that the ferroptosis inducers FIN56, imidazole ketone erastin, and piperazine erastin are substrates of P-glycoprotein. ML-162, GPX inhibitor 26a, and PACMA31 were found to inhibit P-glycoprotein, while GPX inhibitor 26a was additionally able to inhibit ABCG2, suggesting the potential for drug-drug interactions.

pharmacology and toxicology↗

bioRxiv2022

Structure of the SHOC2-MRAS-PP1C complex provides insights into RAF activation and Noonan syndrome

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS and PP1C form a stable ternary complex where all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex only forms when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation, how mutations found in Noonan syndrome enhance the complex formation and reveal new avenues for therapeutic interventions.

biophysics↗

bioRxiv2022

CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature of KRAS mutant cancers

Oncogenic RAS induces perinuclear translocation of the effector kinases ERK and CK2 and their scaffold, KSR1, forming endosomal signaling hubs termed perinuclear signaling centers (PSCs). PSCs are present in all cancer cell lines and tissues examined, suggesting that subcellular compartmentalization of oncogenic kinases drives tumorigenesis. However, the mechanism of perinuclear targeting, whether this location affects kinase substrate specificity, and the importance of PSCs in cancer are unclear. Here we show that the endosomal adaptor, TOLLIP, specifically tethers RAB11A+ signaling endosomes containing CK2 and KSR1 to the perinuclear ER. A predicted {beta}-hairpin fold in TOLLIP mediates binding to the KSR1 CA5 pseudo-kinase domain, recruiting CK2/KSR1 complexes to perinuclear endosomes. TOLLIP is essential for proliferation/survival of tumor cells carrying KRAS and NRAS mutations but not HRAS, BRAF, ERBB or PTEN lesions, or non-transformed cells. KRasG12D-induced lung lesions in Tollip-/- mice displayed reduced numbers of carcinomatous lesions, implicating TOLLIP in malignant progression. TOLLIP-dependent perinuclear CK2 was shown to phosphorylate discrete substrates, including proteins involved in translation and ribosome biogenesis such as RIOK1. Thus, TOLLIP is a key RAS pathway signaling adaptor in K/NRAS tumors whose inhibition is a specific vulnerability of these cancers.

cancer biology↗

bioRxiv2022

Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinantleucine-rich repeat proteins and protein complexes

The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins. HighlightsO_LIImproved yields and purity of LRR proteins and multiprotein complexes through chaperone co-expression, DNA construct design, and use of polycistronic baculovirus. C_LIO_LIOver 300-fold yield increase of SMP protein complex from 0.1 mg/L to 32.6 mg/L. C_LIO_LISUGT1 co-expression is a highly effective technique for recombinant LRR protein expression and purification. C_LIO_LIPolycistronic baculovirus infection is ideal for production of multiprotein complexes C_LI

biochemistry↗

bioRxiv2021

MaveDB v2: a curated community database with over three million variant effects from multiplexed functional assays

A central problem in genomics is understanding the effect of individual DNA variants. Multiplexed Assays of Variant Effect (MAVEs) can help address this challenge by measuring all possible single nucleotide variant effects in a gene or regulatory sequence simultaneously. Here we describe MaveDB v2, which has become the database of record for MAVEs. MaveDB now contains a large fraction of published studies, comprising over two hundred datasets and three million variant effect measurements. We created tools and APIs to streamline data submission and access, transforming MaveDB into a hub for the analysis and dissemination of these impactful datasets.

genomics↗

bioRxiv2021

Camel nanobodies broadly neutralize SARS-CoV-2 variants

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two VHH nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of [≥]5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. One-Sentence SummaryDromedary camel (Camelus dromedarius) VHH phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.

immunology↗

bioRxiv2021

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

synthetic biology↗

bioRxiv2021

Modified E. coli strains enhance baculovirus production by elimination of aberrant transposition events

Baculovirus technology has been the most commonly used expression system for insect cells both due to its potential to generate a large amount of recombinant protein as well as the benefit of post-translational modifications. The most commonly used system to generate recombinant baculoviruses is the Tn7 transposition-based technology known as Bac-to-Bac. Although improvements have been made to this system to further improve quality and reproducibility of baculovirus production, recent data suggests that improved strains still have potential issues with contamination of non-recombinant baculovirus caused by improper transposition into a Tn7 site in the E. coli chromosome. Here we describe a new option for alteration of the E. coli genome to completely block the native Tn7 attachment site, leading to far fewer false positive bacmid colonies being selected and eliminating all risk of non-recombinant baculovirus production.

molecular biology↗

bioRxiv2020

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: O_LIImproved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters C_LIO_LITwo versions of RBD show different sensitivity in serology assays C_LIO_LIYields of greater than 50 mg/l obtained under optimal conditions C_LIO_LIMagnetic bead purification technology improves throughput of protein production C_LI

biochemistry↗

bioRxiv2020

Live imaging of breast tumors shows macrophage-dependent induction and TMEM-mediated enrichment of cancer stem cells during metastatic dissemination

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. We employed high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. A dramatic enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent [~]>60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch signaling. In breast cancers from patients, the density of TMEM doorways correlates strongly with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites. One Sentence SummaryIntravital imaging reveals macrophage-mediated induction of cancer stem cells in vivo and their dramatic enrichment on dissemination through TMEM doorways.

cancer biology↗

bioRxiv2020

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

A vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

biophysics↗