RESUMO
Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it. This labels proteins for capture, extraction, and identification. In this chapter, we present a toolkit for BioID2 specifically adapted for use in E. coli, exemplified by the chemotaxis protein CheA. We have created plasmids containing BioID2 as expression cassettes for proteins (e.g., CheA) fused to BioID2 at either the N or C terminus, optimized with an 8 × GGS linker. We provide a methodology for expression and verification of CheA-BioID2 fusion proteins in E. coli cells, the in vivo biotinylation of interactors by protein-BioID2 fusions, and extraction and analysis of interacting proteins that have been biotinylated.
Assuntos
Biotinilação , Escherichia coli , Mapeamento de Interação de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Biotina/metabolismo , Mapas de Interação de Proteínas , Coloração e Rotulagem/métodos , Plasmídeos/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases/genéticaRESUMO
Anaplastic lymphoma kinase (ALK) is a well-known oncogene involved in various malignancies such as anaplastic large cell lymphoma, lung cancer and neuroblastoma. Several substrates for fused ALK have been identified and their biological functions have been described. However, the lack of a comprehensive identification of ALK substrates limits our understanding of the biological roles of receptor ALK. Thus, this study aimed to identify novel ALK substrates and characterize their biological functions. We screened the interactors of the kinase domain of receptor ALK using proximity-dependent biotin identification and identified 43 interactors. We narrowed down the candidates by evaluating whether these interactors were downstream of ALK in a neuroblastoma cell line, NB-1. Among these, we identified amyloid beta precursor protein binding family B member 1 (APBB1) as an ALK downstream molecule involved in NB-1 cell viability. Finally, we assessed the kinase-substrate relationship between ALK and APBB1 and found that ALK phosphorylated multiple tyrosine residues in APBB1 both in-cell and in-tube assays, with tyrosine 269 as a major target. In conclusion, we successfully identified a new substrate for receptor ALK. Our results may help further elucidate the molecular mechanism of ALK downstream signaling in neuroblastoma.
RESUMO
The Hippo pathway transducers yes-associated protein (YAP) and WW-domain containing transcription regulator 1 (WWTR1/TAZ) are key regulators of liver tumorigenesis, promoting tumor formation and progression. Although the first inhibitors are in clinical trials, targeting the relevant upstream regulators of YAP/TAZ activity could prove equally beneficial. To identify regulators of YAP/TAZ activity in hepatocarcinoma (HCC) cells, we carried out a proximity labelling approach (BioID) coupled with mass spectrometry. We verified CRK-like proto-oncogene adaptor protein (CRKL) as a new YAP-exclusive interaction partner. CRKL is highly expressed in HCC patients, and its expression is associated with YAP activity as well as poor survival prognosis. In vitro experiments demonstrated CRKL-dependent cell survival and the loss of YAP binding induced through actin disruption. Moreover, we delineated the activation of the JNK/JUN pathway by CRKL, which promoted YAP transcription. Our data illustrate that CRKL not only promoted YAP activity through its binding but also through the induction of YAP transcription by JNK/JUN activation. This emphasizes the potential use of targeting the JNK/JUN pathway to suppress YAP expression in HCC patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Nucleares , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proto-Oncogene Mas , Linhagem Celular Tumoral , Ligação Proteica , Sistema de Sinalização das MAP Quinases , Regulação Neoplásica da Expressão Gênica , Transdução de SinaisRESUMO
Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.
RESUMO
Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.
Assuntos
Técnicas de Cultura de Células , Proteína de Morte Celular Associada a bcl , Humanos , Proteína de Morte Celular Associada a bcl/metabolismo , Técnicas de Cultura de Células/métodos , Mapas de Interação de Proteínas , Matriz Extracelular/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas 14-3-3/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Carbono-Nitrogênio Ligases/genética , Ligação Proteica , Proteína bcl-X/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas RepressorasRESUMO
Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.
Assuntos
Biotina , Humanos , Biotina/metabolismo , Zixina/metabolismo , Zixina/genética , Animais , Linhagem Celular , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/fisiologia , Interações Hospedeiro-Patógeno , Alphaherpesvirinae/genética , Alphaherpesvirinae/metabolismo , Sistemas CRISPR-Cas , Células Epiteliais/virologia , Células Epiteliais/metabolismoRESUMO
Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.
Assuntos
Encéfalo , Dependovirus , Proteoma , Proteômica , Espectrometria de Massas em Tandem , Dependovirus/genética , Dependovirus/metabolismo , Animais , Camundongos , Proteômica/métodos , Proteoma/análise , Encéfalo/metabolismo , Espectrometria de Massas em Tandem/métodos , Análise de Célula Única/métodos , Neurônios/metabolismo , Cromatografia Líquida/métodos , Vetores Genéticos/genética , Biotinilação , Espectrometria de Massas/métodos , Astrócitos/metabolismoRESUMO
Formin Homology Proteins (Formins) are a highly conserved family of cytoskeletal regulatory proteins that participate in a diverse range of cellular processes. FMNL2 is a member of the Diaphanous-Related Formin sub-group, and previous reports suggest FMNL2's role in filopodia assembly, force generation at lamellipodia, subcellular trafficking, cell-cell junction assembly, and focal adhesion formation. How FMNL2 is recruited to these sites of action is not well understood. To shed light on how FMNL2 activity is partitioned between subcellular locations, we used biotin proximity labeling and proteomic analysis to identify an FMNL2 interactome. The interactome identified known and new FMNL2 interacting proteins with functions related to previously described FMNL2 activities. In addition, our interactome predicts a novel connection between FMNL2 and extracellular vesicle assembly. We show directly that FMNL2 protein is present in exosomes.
Assuntos
Forminas , Forminas/metabolismo , Humanos , Proteômica/métodos , Exossomos/metabolismo , Espectrometria de Massas/métodos , Ligação Proteica , Células HEK293 , Mapas de Interação de ProteínasRESUMO
Theileria annulata is a tick-transmitted apicomplexan parasite that gained the unique ability among parasitic eukaryotes to transform its host cell, inducing a fatal cancer-like disease in cattle. Understanding the mechanistic interplay between the host cell and malignant Theileria species that drives this transformation requires the identification of responsible parasite effector proteins. In this study, we used TurboID-based proximity labeling, which unbiasedly identified secreted parasite proteins within host cell compartments. By fusing TurboID to nuclear export or localization signals, we biotinylated proteins in the vicinity of the ligase enzyme in the nucleus or cytoplasm of infected macrophages, followed by mass spectrometry analysis. Our approach revealed with high confidence nine nuclear and four cytosolic candidate parasite proteins within the host cell compartments, eight of which had no orthologs in non-transforming T. orientalis. Strikingly, all eight of these proteins are predicted to be highly intrinsically disordered proteins. We discovered a novel tandem arrayed protein family, nuclear intrinsically disordered proteins (NIDP) 1-4, featuring diverse functions predicted by conserved protein domains. Particularly, NIDP2 exhibited a biphasic host cell-cycle-dependent localization, interacting with the EB1/CD2AP/CLASP1 parasite membrane complex at the schizont surface and the tumor suppressor stromal antigen 2 (STAG2), a cohesion complex subunit, in the host nucleus. In addition to STAG2, numerous NIDP2-associated host nuclear proteins implicated in various cancers were identified, shedding light on the potential role of the T. annulata exported protein family NIDP in host cell transformation and cancer-related pathways.IMPORTANCETurboID proximity labeling was used to identify secreted proteins of Theileria annulata, an apicomplexan parasite responsible for a fatal, proliferative disorder in cattle that represents a significant socio-economic burden in North Africa, central Asia, and India. Our investigation has provided important insights into the unique host-parasite interaction, revealing secreted parasite proteins characterized by intrinsically disordered protein structures. Remarkably, these proteins are conspicuously absent in non-transforming Theileria species, strongly suggesting their central role in the transformative processes within host cells. Our study identified a novel tandem arrayed protein family, with nuclear intrinsically disordered protein 2 emerging as a central player interacting with established tumor genes. Significantly, this work represents the first unbiased screening for exported proteins in Theileria and contributes essential insights into the molecular intricacies behind the malignant transformation of immune cells.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas de Protozoários , Theileria annulata , Theileria annulata/genética , Theileria annulata/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Animais , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Bovinos , Interações Hospedeiro-Parasita , Macrófagos/parasitologia , Theileriose/parasitologia , Theileriose/metabolismo , Núcleo Celular/metabolismoRESUMO
Focal adhesions serve as structural and signaling hubs, facilitating bidirectional communication at the cell-extracellular matrix interface. Paxillin and the related Hic-5 (TGFß1i1) are adaptor/scaffold proteins that recruit numerous structural and regulatory proteins to focal adhesions, where they perform both overlapping and discrete functions. In this study, paxillin and Hic-5 were expressed in U2OS osteosarcoma cells as biotin ligase (BioID2) fusion proteins and used as bait proteins for proximity-dependent biotinylation in order to directly compare their respective interactomes. The fusion proteins localized to both focal adhesions and the centrosome, resulting in biotinylation of components of each of these structures. Biotinylated proteins were purified and analyzed by mass spectrometry. The list of proximity interactors for paxillin and Hic-5 comprised numerous shared core focal adhesion proteins that likely contribute to their similar functions in cell adhesion and migration, as well as proteins unique to paxillin and Hic-5 that have been previously localized to focal adhesions, the centrosome, or the nucleus. Western blotting confirmed biotinylation and enrichment of FAK and vinculin, known interactors of Hic-5 and paxillin, as well as several potentially unique proximity interactors of Hic-5 and paxillin, including septin 7 and ponsin, respectively. Further investigation into the functional relationship between the unique interactors and Hic-5 or paxillin may yield novel insights into their distinct roles in cell migration.
RESUMO
Friedreich's Ataxia (FRDA) is a neuromuscular degenerative disorder caused by trinucleotide expansions in the first intron of the frataxin (FXN) gene, resulting in insufficient levels of functional FNX protein. Deficits in FXN involve mitochondrial disruptions including iron-sulfur cluster synthesis and impaired energetics. These studies were to identify unique protein-protein interactions with FXN to better understand its function and design therapeutics. Two complementary approaches were employed, BioID and Co-IP, to identify protein interactions with FXN at the direct binding, indirect binding, and non-proximal levels. Forty-one novel protein interactions were identified by BioID and IP techniques. The FXN protein landscape was further analyzed incorporating both interaction type and functional pathways using a maximum path of 6 proteins with a potential direct interaction between FXN and NFS1. Probing the intersection between FXN-protein landscape and biological pathways associated with FRDA, we identified 41 proteins of interest. Peroxiredoxin 3 (Prdx3) was chosen for further analysis because of its role in mitochondrial oxidative injury. Our data has demonstrated the strengths of employing complementary methods to identify a unique interactome for FXN. Our data provides new insights into FXN function and regulation, a potential direct interaction between FXN and NFS1, and pathway interactions between FXN and Prdx3.
RESUMO
Synapses play a pivotal role in forming neural circuits, with critical implications for brain functions such as learning, memory, and emotions. Several advances in synaptic research have demonstrated the diversity of synaptic structure and function, which can form thousands of connections depending on the neuronal cell types. Moreover, synapses not only interconnect neurons but also establish connections with glial cells such as astrocytes, which play a key role in the architecture and function of neuronal circuits in the brain. Emerging evidence suggests that dysfunction of synaptic proteins contributes to a variety of neurological and psychiatric disorders. Therefore, it is crucial to determine the molecular networks within synapses in various neuronal cell types to gain a deeper understanding of how the nervous system regulates brain function. Recent advances in synaptic proteome approaches, such as fluorescence-activated synaptosome sorting (FASS) and proximity labeling, have allowed for a detailed and spatial analysis of many cell-type-specific synaptic molecules in vivo. In this brief review, we highlight these novel spatial proteomic approaches and discuss the regulation of synaptic formation and function in the brain. This knowledge of molecular networks provides new insight into the understanding of many neurological and psychiatric disorders.
RESUMO
ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13, regulates the length of Von Willebrand factor (VWF) multimers and their platelet-binding activity. ADAMTS13 is constitutively secreted as an active protease and is not inhibited by circulating protease inhibitors. Therefore, the mechanisms that regulate ADAMTS13 protease activity are unknown. We performed an unbiased proteomics screen to identify ligands of ADAMTS13 by optimizing the application of BioID to plasma. Plasma BioID identified 5 plasma proteins significantly labeled by the ADAMTS13-birA* fusion, including VWF and plasminogen. Glu-plasminogen, Lys-plasminogen, mini-plasminogen, and apo(a) bound ADAMTS13 with high affinity, whereas micro-plasminogen did not. None of the plasminogen variants or apo(a) bound to a C-terminal truncation variant of ADAMTS13 (MDTCS). The binding of plasminogen to ADAMTS13 was attenuated by tranexamic acid or ε-aminocaproic acid, and tranexamic acid protected ADAMTS13 from plasmin degradation. These data demonstrate that plasminogen is an important ligand of ADAMTS13 in plasma by binding to the C-terminus of ADAMTS13. Plasmin proteolytically degrades ADAMTS13 in a lysine-dependent manner, which may contribute to its regulation. Adapting BioID to identify protein-interaction networks in plasma provides a powerful new tool to study protease regulation in the cardiovascular system.
Assuntos
Fibrinolisina , Ácido Tranexâmico , Fibrinolisina/metabolismo , Fator de von Willebrand/metabolismo , Proteína ADAMTS13 , Proteínas ADAM/metabolismo , Ligantes , Plasminogênio/metabolismoRESUMO
DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.
Assuntos
Replicação do DNA , Hidroxiureia , Proteômica , Hidroxiureia/farmacologia , Proteômica/métodos , Humanos , Dano ao DNA , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteoma/metabolismoRESUMO
KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.
Assuntos
Genes ras , Proteínas Proto-Oncogênicas p21(ras) , Animais , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/genética , Alelos , Biotina/química , Estreptavidina , MutaçãoRESUMO
The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified â¼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.
Assuntos
Fatores de Ribosilação do ADP , Fosfolipase D , Transdução de Sinais , Fatores de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Humanos , Fosfolipase D/metabolismo , Fosfolipase D/genética , Células HEK293 , Animais , Nexinas de Classificação/metabolismo , Nexinas de Classificação/genética , Mapeamento de Interação de ProteínasRESUMO
Borrowing some quotes from Harper Lee's novel "To Kill A Mockingbird" to help frame our manuscript, we discuss methods to profile local proteomes. We initially focus on chemical biology regimens that function in live organisms and use reactive biotin species for this purpose. We then consider ways to add new dimensions to these experimental regimens, principally by releasing less reactive (i. e., more selective) (preter)natural electrophiles. Although electrophile release methods may have lower resolution and label fewer proteins than biotinylation methods, their ability to probe simultaneously protein function and locale raises new and interesting possibilities for the field.
Assuntos
Biotina , Proteoma , BiotinilaçãoRESUMO
Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.
Assuntos
Biotina , Proteínas de Ligação a DNA , Biotinilação , Estreptavidina/química , Sefarose , Tripsina , Biotina/químicaRESUMO
The mammalian cell is a complex entity, with membrane-bound and membrane-less organelles playing vital roles in regulating cellular homeostasis. Organellar protein niches drive discrete biological processes and cell functions, thus maintaining cell equilibrium. Cellular processes such as signaling, growth, proliferation, motility, and programmed cell death require dynamic protein movements between cell compartments. Aberrant protein localization is associated with a wide range of diseases. Therefore, analyzing the subcellular proteome of the cell can provide a comprehensive overview of cellular biology. With recent advancements in mass spectrometry, imaging technology, computational tools, and deep machine learning algorithms, studies pertaining to subcellular protein localization and their dynamic distributions are gaining momentum. These studies reveal changing interaction networks because of "moonlighting proteins" and serve as a discovery tool for disease network mechanisms. Consequently, this review aims to provide a comprehensive repository for recent advancements in subcellular proteomics subcontexting methods, challenges, and future perspectives for method developers. In summary, subcellular proteomics is crucial to the understanding of the fundamental cellular mechanisms and the associated diseases.