Development and Validation of a Proximity Labeling Fusion Protein Construct to Identify the Protein-Protein Interactions of Transcription Factors.

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.

Real-time analysis of the biomolecular interaction between gelsolin and Aß1-42 monomer and its implication for Alzheimer's disease.

Biomolecular interaction acts a pivotal part in understanding the mechanisms underlying the development of Alzheimer's disease (AD). Herein, we built a biosensing platform to explore the interaction between gelsolin (GSN) and different ß-amyloid protein 1-42 (Aß1-42) species, including Aß1-42 monomer (m-Aß), Aß1-42 oligomers with both low and high levels of aggregation (LLo-Aß and HLo-Aß) via dual polarization interferometry (DPI). Real-time molecular interaction process and kinetic analysis showed that m-Aß had the strongest affinity and specificity with GSN compared with LLo-Aß and HLo-Aß. The impact of GSN on inhibiting aggregation of Aß1-42 and solubilizing Aß1-42 aggregates was evaluated by circular dichroism (CD) spectroscopy. The maintenance of random coil structure of m-Aß and the reversal of ß-sheet structure in HLo-Aß were observed, demonstrating the beneficial effects of GSN on preventing Aß from aggregation. In addition, the structure of m-Aß/GSN complex was analyzed in detail by molecular dynamics (MD) simulation and molecular docking. The specific binding sites and crucial intermolecular forces were identified, which are believed to stabilize m-Aß in its soluble state and to inhibit the fibrilization of Aß1-42. Combined theoretical simulations and experiment results, we speculate that the success of GSN sequestration mechanism and the balance of GSN levels in cerebrospinal fluid and plasma of AD subjects may contribute to a delay in AD progression. This research not only unveils the molecular basis of the interaction between GSN and Aß1-42, but also provides clues to understanding the crucial functions of GSN in AD and drives the development of AD drugs and therapeutic approaches.

The actin-binding protein palladin associates with the respiratory syncytial virus matrix protein.

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescent-tagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release.IMPORTANCERespiratory syncytial virus is responsible for severe lower respiratory tract infections in young children under 5 years old, the elderly, and the immunosuppressed. The interaction of the respiratory syncytial virus matrix protein with the host actin cytoskeleton is important in infection but has not been investigated in depth. In this study, we show that the respiratory syncytial virus matrix protein associates with actin microfilaments and the actin-binding protein palladin, suggesting a role for palladin in respiratory syncytial virus release. This study provides new insight into the role of the actin cytoskeleton in respiratory syncytial virus infection, a key host-RSV interaction in assembly. Understanding the mechanism by which the RSV M protein and actin interact will ultimately provide a basis for the development of therapeutics targeted at RSV infections.

Neuroinflammation inhibition and neuroprotective effects of purpurin, a potential anti-AD compound, screened via network proximity and gene enrichment analyses.

Alzheimer's disease (AD) is a complex neurodegenerative disease without any effective preventive or therapeutic drugs. Natural products with stable structures and pharmacological characteristics are valuable sources for the development of novel drugs for many complex diseases. This study aimed to discover potential natural compounds for the treatment of AD using new technologies and methods and explore the efficacy and mechanism of candidate compounds. AD-related large-scale genetic datasets were collated to construct disease-PPIs and natural products were collected from six databases to construct compound-protein interactions (CPIs). Potential relationships between natural compounds and AD were predicted via network proximity and gene enrichment analyses. Then, five AD-related cell models and d-galactose-induced aging rat model were established to evaluate the neuroprotective effects of candidate compounds in vitro and in vivo. We identified that 267 natural compounds were predicted to have close connections with AD and 19 compounds could exert protective effect in at least one cell model. Notably, purpurin exerted protective effect in three cell models and significantly improved the cognitive learning and memory functions, reduced the oxidative stress injuries and neuroinflammation, and enhanced the synaptic plasticity and neurotrophic effect in the brain of d-galactose-treated rats. In this study, AD-related natural compounds were identified via network proximity and gene enrichment analyses. In vivo and in vitro experiments revealed the therapeutic potential of purpurin for AD treatment, laying the foundation for further in-depth research and providing valuable information for the development of novel anti-AD drugs.

Identification of function modules in the co-expression protein-protein interaction network of Bombyx mori in response to Beauveria bassiana infection.

Beauveria bassiana (B. bassiana) is a common fungal disease in sericulture. Previous research has primarily focused on investigating genes involved in innate immunity. However, the response of Bombyx mori (B. mori) to B. bassiana requires the coordination of other biological processes in addition to the immune system. We measured protein expression profile of B. mori after inoculating B. bassiana using iTRAQ technology in previous. Here we constructed a co-expression protein-protein interaction network of B. mori in response to B. bassiana infection. Subnetworks and modules were analyzed, and the functions of these modules were annotated. The results revealed the identification of numerous proteins associated with cellular immunity, including those involved in phagosomes, lysosomes, mTOR signaling, sugar metabolism, and the ubiquitin-proteasome pathway. Meanwhile, we observed that the pathways involved in protein synthesis were activated, including pyruvate and purine metabolism, RNA transport, ribosome, protein processing in endoplasmic reticulum, and protein export pathways, during B. bassiana infection. Based on this analysis, we selected six candidate genes (shock protein, ribosome, translocon, actin muscle-type A2, peptidoglycan recognition protein, and collagenase) that were found to be related to the response to B. bassiana. Further verification experiments demonstrated significant changes in their expression levels after inoculation with B. bassiana. These research findings provide new insights into the molecular mechanism of insect immune response to fungal infection.

Profiling Cullin4-E3 ligases interactomes and their rewiring in influenza A virus infection.

Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.

In-silico analysis predicts disruption of normal angiogenesis as a causative factor in osteoporosis pathogenesis.

Angiogenesis-osteogenesis coupling is critical for proper functioning and maintaining the health of bones. Any disruption in this coupling, associated with aging and disease, might lead to loss of bone mass. Osteoporosis (OP) is a debilitating bone metabolic disorder that affects the microarchitecture of bones, gradually leading to fracture. Computational analysis revealed that normal angiogenesis is disrupted during the progression of OP, especially postmenopausal osteoporosis (PMOP). The genes associated with OP and PMOP were retrieved from the DisGeNET database. Hub gene analysis and molecular pathway enrichment were performed via the Cytoscape plugins STRING, MCODE, CytoHubba, ClueGO and the web-based tool Enrichr. Twenty-eight (28) hub genes were identified, eight of which were transcription factors (HIF1A, JUN, TP53, ESR1, MYC, PPARG, RUNX2 and SOX9). Analysis of SNPs associated with hub genes via the gnomAD, I-Mutant2.0, MUpro, ConSurf and COACH servers revealed the substitution F201L in IL6 as the most deleterious. The IL6 protein was modeled in the SWISS-MODEL server and the substitution was analyzed via the YASARA FoldX plugin. A positive ΔΔG (1.936) of the F201L mutant indicates that the mutated structure is less stable than the wild-type structure is. Thirteen hub genes, including IL6 and the enriched molecular pathways were found to be profoundly involved in angiogenesis/endothelial function and immune signaling. Mechanical loading of bones through weight-bearing exercises can activate osteoblasts via mechanotransduction leading to increased bone formation. The present study suggests proper mechanical loading of bone as a preventive strategy for PMOP, by which angiogenesis and the immune status of the bone can be maintained. This in silico analysis could be used to understand the molecular etiology of OP and to develop novel therapeutic approaches.

Progress in mass spectrometry approaches to profiling protein-protein interactions in the studies of the innate immune system.

Understanding protein-protein interactions (PPIs) is pivotal for deciphering the intricacies of biological processes. Dysregulation of PPIs underlies a spectrum of diseases, including cancer, neurodegenerative disorders, and autoimmune conditions, highlighting the imperative of investigating these interactions for therapeutic advancements. This review delves into the realm of mass spectrometry-based techniques for elucidating PPIs and their profound implications in biological research. Mass spectrometry in the PPI research field not only facilitates the evaluation of protein-protein interaction modulators but also discovers unclear molecular mechanisms and sheds light on both on- and off-target effects, thus aiding in drug development. Our discussion navigates through six pivotal techniques: affinity purification mass spectrometry (AP-MS), proximity labeling mass spectrometry (PL-MS), cross-linking mass spectrometry (XL-MS), size exclusion chromatography coupled with mass spectrometry (SEC-MS), limited proteolysis-coupled mass spectrometry (LiP-MS), and thermal proteome profiling (TPP).

Probing hot spots of protein-protein interactions mediated by the safety-belt region of REV7.

REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors.

Predicting protein interactions of the kinase Lck critical to T cell modulation.

Protein-protein interactions (PPIs) play pivotal roles in directing T cell fate. One key player is the non-receptor tyrosine protein kinase Lck that helps to transduce T cell activation signals. Lck is mediated by other proteins via interactions that are inadequately understood. Here, we use the deep learning method AF2Complex to predict PPIs involving Lck, by screening it against ∼1,000 proteins implicated in immune responses, followed by extensive structural modeling for selected interactions. Remarkably, we describe how Lck may be specifically targeted by a palmitoyltransferase using a phosphotyrosine motif. We uncover "hotspot" interactions between Lck and the tyrosine phosphatase CD45, leading to a significant conformational shift of Lck for activation. Lastly, we present intriguing interactions between the phosphotyrosine-binding domain of Lck and the cytoplasmic tail of the immune checkpoint LAG3 and propose a molecular mechanism for its inhibitory role. Together, this multifaceted study provides valuable insights into T cell regulation and signaling.

The Effect of Vitamin D Deficiency on Immune-Related Hub Genes: A Network Analysis Associated With Type 1 Diabetes.

Background Type 1 diabetes (T1D) is an autoimmune disorder that results in the destruction of pancreatic beta cells, causing a shortage of insulin secretion. The development of T1D is influenced by both genetic predisposition and environmental factors, such as vitamin D. This vitamin is known for its ability to regulate the immune system and has been associated with a decreased risk of T1D. However, the specific ways in which vitamin D affects immune regulation and the preservation of beta cells in T1D are not yet fully understood. Gaining a better understanding of these interactions is essential for identifying potential targets for preventing and treating T1D. Methods The analysis focused on two Gene Expression Omnibus (GEO) datasets, namely, GSE55098 and GSE50012, to detect differentially expressed genes (DEGs). Enrichr (Ma'ayan Laboratory, New York, NY) was used to perform enrichment analysis for the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The Search Tool for the Retrieval of Interacting Genes 12.0 (STRING) database was used to generate a protein-protein interaction (PPI) network. The Cytoscape 3.10.1 (Cytoscape Team, San Diego, CA) was used to analyze the PPI network and discover the hub genes. Results The DEGs in both datasets were identified using the GEO2R tool, with a particular focus on genes exhibiting contrasting regulations. Enrichment analysis unveiled the participation of these oppositely regulated DEGs in processes relevant to the immune system. Cytoscape analysis of the PPI network revealed five hub genes, MNDA, LILRB2, FPR2, HCK, and FCGR2A, suggesting their potential role in the pathogenesis of T1D and the response to vitamin D. Conclusion The study elucidates the complex interaction between vitamin D metabolism and immune regulation in T1D. The identified hub genes provide important knowledge on the molecular pathways that underlie T1D and have the potential to be targeted for therapeutic intervention. This research underscores the importance of vitamin D in the immune system's modulation and its impact on T1D development.

Oxidative protein damage negatively affects protein-protein interaction: The case of KRAS-cRAF.

Protein-protein interactions (PPIs) play crucial roles in cellular signaling, transmitting signals from the cell surface to its interior. One of the most important signaling cascades is the RAS-RAF-MEK-ERK pathway. This pathway is initiated by various upstream signaling reactions, including receptor tyrosine kinase (RTK) activation, and it controls many biological functions like cell proliferation, differentiation, and survival. Once RAS is activated, it binds RAF and relays the signal to downstream proteins. The RAS-binding domain (RBD) in RAF protein plays a crucial role in this process, facilitating the RAS-ERK pathway signaling. In this study, we explored the effect of oxidative stress induced by UV radiation on the KRAS-RBD interaction. Using the Split Intein-Mediated Protein Ligation (SIMPL) method, we assessed the impact of different UV doses on KRAS-RBD interactions and observed a disruption of this interaction at higher doses. UV-treated samples exhibited high levels of protein carbonylation, as detected by Oxime Blot and mass spectrometry (MS) analysis, indicating oxidative damage. The MS results provided detailed insights into specific carbonylation modifications on the KRAS protein. Our study demonstrates that protein oxidation and carbonylation can disrupt protein-protein interactions, specifically the KRAS/c-RAF interaction. These findings highlight the impact of oxidative stress on signaling pathways, such as those triggered by UV irradiation. A deeper understanding of these molecular changes may aid in developing therapies targeting diseases linked to oxidative stress, including cancer.

Understanding of formation, gastrointestinal breakdown, and application of whey protein emulsion gels: Insights from intermolecular interactions.

Whey protein emulsion gel is an ideal model food for revealing how the multilength scale food structures affect food digestion, as their structure and mechanical properties can be precisely manipulated by controlling the type and intensity of intermolecular interactions between protein molecules. However, there are still significant understanding gaps among intermolecular interactions, protein aggregation and gelation, emulsion gel formation, gel breakdown in the gastrointestinal tract (GIT), and the practical use of whey protein emulsion gels, which limits their GIT-targeted applications. In this regard, the relationship between the structure and digestion behavior of heat-set whey protein emulsion gels is reviewed and discussed mainly from the following aspects: (1) structural characteristics of whey protein molecules; (2) how different types of intermolecular interactions influence heat-induced aggregation and gelation of whey protein in the aqueous solutions and the oil-in-water emulsions, and the mechanical properties of the final gels; (3) functions of the mouth, the stomach, and the small intestine in processing of solid foods, and how different types of intermolecular interactions influence the breakdown properties of heat-set whey protein emulsion gels in GIT (i.e., their respective role in controlling gel digestion). Finally, the implications of knowledge derived from the formation and gastrointestinal breakdown of heat-set whey protein emulsion gels for developing controlled delivery vehicles, human satiety enhancers, and sensory modifiers are highlighted.

S-CDK-regulated bipartite interaction of Mcm10 with MCM is essential for DNA replication.

Mcm10 plays an essential role in the activation of replicative helicase CMG through the cell cycle-regulated interaction with the prototype MCM double hexamer in Saccharomyces cerevisiae. In this study, we reported that Mcm10 is phosphorylated by S-phase cyclin-dependent kinases (S-CDKs) at S66, which enhances Mcm10--MCM association during the S phase. S66A single mutation or even deletion of whole N-terminus (a.a. 1-128) only causes mild growth defects. Nevertheless, S66 becomes indispensable in the absence of the Mcm10 C-terminus ((a.a. 463-571), the major MCM-binding domain. Using a two-degron strategy to efficiently deplete Mcm10, we show that mcm10-S66AΔC has a severe defect in proceeding into the S phase. Notably, both lethality and S-phase deficiency can be rescued by artificially tethering mcm10-S66AΔC to MCM. These findings illustrate how the Mcm10-MCM association is regulated as a crucial event in DNA replication initiation.

Identification and validation of hub differential genes in pulmonary sarcoidosis.

A total of 138 cDEGs were screened from mediastinal lymph nodes and peripheral whole blood. Among them, 6 hub cDEGs including CTSS, CYBB, FPR2, MNDA, TLR1 and TLR8 with elevated degree and betweenness levels were illustrated in protein-protein interaction network. In comparison to healthy controls, CTSS (1.61 vs. 1.05), CYBB (1.68 vs. 1.07), FPR2 (2.77 vs. 0.96), MNDA (2.14 vs. 1.23), TLR1 (1.56 vs. 1.09), and TLR8 (2.14 vs. 0.98) displayed notably elevated expression levels within pulmonary sarcoidosis PBMC samples (P < 0.0001 for FPR2 and P < 0.05 for others), echoing with prior mRNA microarray findings. The most significant functional pathways were immune response, inflammatory response, plasma membrane and extracellular exosome, with 6 hub cDEGs distributing along these pathways. CTSS, CYBB, FPR2, MNDA, TLR1, and TLR8 could be conducive to improving the diagnostic process and understanding the underlying mechanisms of pulmonary sarcoidosis.

Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR.

Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

Temperature-jump microscopy and interaction of Hsp70 heat shock protein with a client protein in vivo.

Biomolecular processes such as protein-protein interactions can depend strongly on cell type and even vary within a single cell type. Here we develop a microscope with a Peltier-controlled temperature stage, a laser temperature jump to induce heat stress, and an autofocusing feature to mitigate temperature drift during experiments, to study a protein-protein interaction in a selected cell type within a live organism, the zebrafish larva. As an application of the instrument, we show that there is considerable cell-to-cell variation of the heat shock protein Hsp70 binding to one of its clients, phosphoglycerate kinase in vivo. We adapt a key feature from our previous folding study, rare transformation of cells within the larva, so that individual cells can be imaged and differentiated for cell-to-cell response. Our approach can be extended to other organisms and cell types than the ones demonstrated in this work.

Interaction of dairy and plant proteins for improving the emulsifying and gelation properties in food matrices: a review.

A variety of variables influence food texture, two of which are gelation and emulsification. Protein interactions have an important role in influencing gelation and emulsifying properties. The utilization of plant proteins in the development of food systems is a prominent subject within the current protein transition paradigm. Plant proteins diminish gel strength compared to dairy proteins. Protein providers prefer to create their own networks rather than rely on tight ties. It may be feasible to resolve these challenges if the interactions between plant and dairy proteins are known at all sizes, from molecular to macroscopic. Therefore, the proteins and dairy proteins are the main emphasis of this review. The role of these proteins in interacting with food matrices is also discussed. Additionally, this data gives information on worldwide research trends. Finally, a glimpse into the future was discussed.

Target-templated Construction of Functional Proteomimetics Using Photo-foldamer Libraries.

Current methods for proteomimetic engineering rely on structure-based design. Here we describe a design strategy that allows the construction of proteomimetics against challenging targets without a priori characterization of the target surface. Our approach relies on (i) a 100-membered photoreactive foldamer library, the members of which act as local surface mimetics, and (ii) the subsequent affinity maturation of the primary hits using systems chemistry. Two surface-oriented proteinogenic side chains drove the interactions between the short helical foldamer fragments and the proteins. Diazirine-based photo-crosslinking was applied to sensitively detected and localize binding even to shallow and dynamic patches on representatively difficult targets. Photo-foldamers identified functionally relevant protein interfaces, allosteric and previously unexplored targetable regions on the surface of STAT3 and an oncogenic K-Ras variant. Target-templated dynamic linking of foldamer hits resulted in two orders of magnitude affinity improvement in a single step. The dimeric K-Ras ligand mimicked protein-like catalytic functions. The photo-foldamer approach thus enables the highly efficient mapping of protein-protein interaction sites and provides a viable starting point for proteomimetic ligand development without a priori structural hypotheses.

Peptide inhibitors targeting Ras and Ras-associated protein-protein interactions.

Peptides represent attractive molecules for targeting protein-protein interactions, and peptide drug development has made great progress during the last decades. Ras protein, the most promising target in cancer therapy, is one of the major growth drivers in various cancers. Although many small molecule inhibitors have been reported to effectively target Ras protein and some inhibitors (such as MRTX849 and AMG 510) have been translated into clinical application, just a few peptide inhibitors have been reported. Here we summarize different types of peptide inhibitors, including monocyclic peptides, bicyclic peptides, stapled peptides, and proteomimetic inhibitors, developed in recent years; emphasize the limits and achievements; and discuss the outlook and challenges associated with future research in peptide inhibitors. This review aims to provide a reference for the discovery of Ras peptide inhibitors.