Protein Crosstalk Research Articles

Innentitelbild: Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners (Angew. Chem. 7/2024)

Innentitelbild: Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners (Angew. Chem. 7/2024)

Inside Cover: Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners (Angew. Chem. Int. Ed. 7/2024)

Inside Cover: Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners (Angew. Chem. Int. Ed. 7/2024)

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners

AbstractDrawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non‐covalently linked, we have devised a method to establish unnatural, effector‐mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK‐3) through non‐covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK‐3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK‐3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell‘s hypoxic response. These findings demonstrate a step toward imitating the function of effector‐regulated cell‐signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof‐of‐principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell‘s environment and consequent protein expression profile.

Cell polarity: Adapting the PAR cascade to diverse cellular contexts

Two new studies shed light on the intricacies of Caenorhabditis elegans embryo patterning, revealing how the conserved interaction and crosstalk of PAR proteins are adapted to perceive distinct cues, ultimately shaping unique asymmetries in form and function.

Cell-in-cell structure in cancer: evading strategies from anti-cancer therapies.

One of the regulated forms of cell death is the cell-in-cell (CIC) structure, in which a surviving cell is engulfed by another cell, a mechanism that causes the death of the engulfed cell by an adjacent cell. Several investigators have previously shown that the presence of CICs is an independent risk factor significantly associated with decreased survival in patients with various types of cancer. In this review, we summarize the role of CIC in the tumor microenvironment (TME), including changes and crosstalk of molecules and proteins in the surrounding CIC, and the role of these factors in contributing to therapeutic resistance acquisition. Moreover, CIC structure formation is influenced by the modulation of TME, which may lead to changes in cellular properties. Future use of CIC as a clinical diagnostic tool will require a better understanding of the effects of chemotherapy on CIC, biomarkers for each CIC formation process, and the development of automated CIC detection methods in tissue sections of tumor specimens.

Integrin β3 directly inhibits the Gα13-p115RhoGEF interaction to regulate G protein signaling and platelet exocytosis

The integrins and G protein-coupled receptors are both fundamental in cell biology. The cross talk between these two, however, is unclear. Here we show that β3 integrins negatively regulate G protein-coupled signaling by directly inhibiting the Gα13-p115RhoGEF interaction. Furthermore, whereas β3 deficiency or integrin antagonists inhibit integrin-dependent platelet aggregation and exocytosis (granule secretion), they enhance G protein-coupled RhoA activation and integrin-independent secretion. In contrast, a β3-derived Gα13-binding peptide or Gα13 knockout inhibits G protein-coupled RhoA activation and both integrin-independent and dependent platelet secretion without affecting primary platelet aggregation. In a mouse model of myocardial ischemia/reperfusion injury in vivo, the β3-derived Gα13-binding peptide inhibits platelet secretion of granule constituents, which exacerbates inflammation and ischemia/reperfusion injury. These data establish crucial integrin-G protein crosstalk, providing a rationale for therapeutic approaches that inhibit exocytosis in platelets and possibly other cells without adverse effects associated with loss of cell adhesion.

S100s and HMGB1 Crosstalk in Pancreatic Cancer Tumors.

Pancreatic cancer remains a disease that is very difficult to treat. S100 proteins are small calcium binding proteins with diverse intra- and extracellular functions that modulate different aspects of tumorigenesis, including tumor growth and metastasis. High mobility group box 1 (HMGB1) protein is a multifaceted protein that also actively influences the development and progression of tumors. In this study, we investigate the possible correlations, at the transcript level, between S100s and HMGB1 in pancreatic cancer. For this purpose, we calculated Pearson's correlations between the transcript levels of 13 cancer-related S100 genes and HMGB1 in a cDNA array containing 19 pancreatic cancer tumor samples, and in 8 human pancreatic cancer cell lines. Statistically significant positive correlations were found in 5.5% (5 out of 91) and 37.4% (34 of 91) of the possible S100/S100 or S100/HMGB1 pairs in cells and tumors, respectively. Our data suggest that many S100 proteins crosstalk in pancreatic tumors either with other members of the S100 family, or with HMGB1. These newly observed interdependencies may be used to further the characterization of pancreatic tumors based on S100 and HMGB1 transcription profiles.

Targeting the Cysteine Redox Proteome in Parkinson's Disease: The Role of Glutathione Precursors and Beyond.

Encouraging recent data on the molecular pathways underlying aging have identified variants and expansions of genes associated with DNA replication and repair, telomere and stem cell maintenance, regulation of the redox microenvironment, and intercellular communication. In addition, cell rejuvenation requires silencing some transcription factors and the activation of pluripotency, indicating that hidden molecular networks must integrate and synchronize all these cellular mechanisms. Therefore, in addition to gene sequence expansions and variations associated with senescence, the optimization of transcriptional regulation and protein crosstalk is essential. The protein cysteinome is crucial in cellular regulation and plays unexpected roles in the aging of complex organisms, which show cumulative somatic mutations, telomere attrition, epigenetic modifications, and oxidative dysregulation, culminating in cellular senescence. The cysteine thiol groups are highly redox-active, allowing high functional versatility as structural disulfides, redox-active disulfides, active-site nucleophiles, proton donors, and metal ligands to participate in multiple regulatory sites in proteins. Also, antioxidant systems control diverse cellular functions, including the transcription machinery, which partially depends on the catalytically active cysteines that can reduce disulfide bonds in numerous target proteins, driving their biological integration. Since we have previously proposed a fundamental role of cysteine-mediated redox deregulation in neurodegeneration, we suggest that cellular rejuvenation of the cysteine redox proteome using GSH precursors, like N-acetyl-cysteine, is an underestimated multitarget therapeutic approach that would be particularly beneficial in Parkinson's disease.

Workflow enabling deepscale immunopeptidome, proteome, ubiquitylome, phosphoproteome, and acetylome analyses of sample-limited tissues

Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each ‘ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.

The crosstalk among autophagy, apoptosis, and pyroptosis in cardiovascular disease.

In recent years, the mechanism of cell death has become a hotspot in research on the pathogenesis and treatment of cardiovascular disease (CVD). Different cell death modes, including autophagy, apoptosis, and pyroptosis, are mosaic with each other and collaboratively regulate the process of CVD. This review summarizes the interaction and crosstalk of key pathways or proteins which play a critical role in the entire process of CVD and explores the specific mechanisms. Furthermore, this paper assesses the interrelationships among these three cell deaths and reviews how they regulate the pathogenesis of CVD. By understanding how these three cell death modes go together we can learn about the pathogenesis of CVD, which will enable us to identify new targets for preventing, controlling, and treating CVD. It will not only reduce mortality but also improve the quality of life.

Protein Kinases and Cross-talk between Post-translational Modifications in the Regulation of Drug Transporters.

Drug transporters are modulators for drug absorption, distribution, and excretion. Key drug transporters including P-glycoprotein and breast cancer resistance protein of the ABC superfamily; organic anion transporting polypeptide 1B1 and 1B3, organic anion transporter 1 and 3, and organic cation transporter 2, as well as multidrug and toxin extrusion 1 and 2 of the SLC superfamily have been recommended by regulatory agencies to be investigated and evaluated in drug-drug interaction (DDI) studies due to their important roles in determining the efficacy, toxicity and DDI of various drugs. Drug transporters are subjected to multiple levels of control and post-translational modifications (PTMs) provide rapid and versatile ways of regulation. Under pathologic and/or pharmacological conditions, PTMs may be altered in the cellular system, leading to functional changes of transporter proteins. Phosphorylation is by far the most actively investigated form of PTMs in the regulation of transporters. Further, studies in recent years also found that protein kinases coordinate with other PTMs for the dynamic control of these membrane proteins. Here we summarized the regulation of major drug transporters by protein kinases and their cross-talking with other PTMs that may generate a complex regulatory network for fine-tuning the function of these important drug processing modulators. SIGNIFICANCE STATEMENT: Kinases regulate drug transporters in versatile manners; Kinase regulation cross-talks with other PTMs, forming a complex network for transporter regulation; Pathological and/or pharmacological conditions may alter PTMs and affect transporter function with different molecular mechanisms.

Implications of critical node-dependent unidirectional cross-talk of Plasmodium SUMO pathway proteins

The endoparasitic pathogen, Plasmodium falciparum (Pf), modulates protein-protein interactions to employ post-translational modifications like SUMOylation to establish successful infections. The interaction between E1 and E2 (Ubc9) enzymes governs species specificity in the Plasmodium SUMOylation pathway. Here, we demonstrate that a unidirectional cross-species interaction exists between Pf-SUMO and human E2, whereas Hs-SUMO1 failed to interact with Pf-E2. Biochemical and biophysical analyses revealed that surface-accessible aspartates of Pf-SUMO determine the efficacy and specificity of SUMO-Ubc9 interactions. Furthermore, we demonstrate that critical residues of the Pf-Ubc9 N terminus are responsible for diminished Hs-SUMO1 and Pf-Ubc9 interaction. Mutating these residues to corresponding Hs-Ubc9 residues restores electrostatic, π−π, and hydrophobic interactions and allows efficient cross-species interactions. We suggest that, in comparison with human counterparts, Plasmodium SUMO and Ubc9 proteins have acquired critical changes on their surfaces as nodes, which Plasmodium can use to exploit the host SUMOylation machinery.

ADP-Ribosylation Factor Family of Small GTP-Binding Proteins: Their Membrane Recruitment, Activation, Crosstalk and Functions.

Members of the ADP-ribosylation factor (ARF) family of guanine-nucleotide binding proteins play critical roles in various cellular processes, especially in regulating the secretory, and endocytic pathways. The fidelity of intracellular vesicular trafficking depends on proper activations and precise subcellular distributions of ARF family proteins regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here we review recent progress in understanding the membrane recruitment, activation, crosstalk, and functions of ARF family proteins.

Exploration of Protein Posttranslational Modification Landscape and Cross Talk with CrossTalkMapper.

Post-translational modifications (PTMs) of proteins play crucial roles in defining protein function. They often do not occur alone, leading to a large variety of proteoforms that correspond to different combinations of multiple PTMs simultaneously decorating a protein. Changes of these proteoforms can be quantified via middle-down and top-down mass spectrometry experiments where the simultaneous PTM settings are obtained by measuring long peptides or entire proteins. Data from such experiments poses big challenges in identifying relevant features of biological and clinical importance. Generally, multiple data layers need to be considered such as proteoforms, individual PTMs, and PTM types. Therein, visualization methods are a crucial part of data analysis as they provide, if applied correctly, insights into both general behaviors as well as a deep view into fine-grained behavior. Here, we present a workflow to visualize histone proteins and their myriad of PTMs based on different R visualization modules applied to data from quantitative middle-down experiments. The procedure can be adapted to diverse experimental designs and is applicable to different proteins and PTMs.

Amyloid-beta peptide and tau protein crosstalk in Alzheimer's disease.

Alzheimer’s disease is a neurodegenerative disease that accounts for most of the 50-million dementia cases worldwide in 2018. A large amount of evidence supports the amyloid cascade hypothesis, which states that amyloid-beta accumulation triggers tau hyperphosphorylation and aggregation in form of neurofibrillary tangles, and these aggregates lead to inflammation, synaptic impairment, neuronal loss, and thus to cognitive decline and behavioral abnormalities. The poor correlation found between cognitive decline and amyloid plaques, have led the scientific community to question whether amyloid-beta accumulation is actually triggering neurodegeneration in Alzheimer’s disease. The occurrence of tau neurofibrillary tangles better correlates to neuronal loss and clinical symptoms and, although amyloid-beta may initiate the cascade of events, tau impairment is likely the effector molecule of neurodegeneration. Recently, it has been shown that amyloid-beta and tau cooperatively work to impair transcription of genes involved in synaptic function and, more importantly, that downregulation of tau partially reverses transcriptional perturbations. Despite mounting evidence points to an interplay between amyloid-beta and tau, some factors could independently affect both pathologies. Thus, the dual pathway hypothesis, which states that there are common upstream triggers causing both amyloid-beta and tau abnormalities has been proposed. Among others, the immune system seems to be strongly involved in amyloid-beta and tau pathologies. Other factors, as the apolipoprotein E ε4 isoform has been suggested to act as a link between amyloid-beta and tau hyperphosphorylation. Interestingly, amyloid-beta-immunotherapy reduces not only amyloid-beta but also tau levels in animal models and in clinical trials. Likewise, it has been shown that tau-immunotherapy also reduces amyloid-beta levels. Thus, even though amyloid-beta immunotherapy is more advanced than tau-immunotherapy, combined amyloid-beta and tau-directed therapies at early stages of the disease have recently been proposed as a strategy to stop the progression of Alzheimer’s disease.

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission.

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

Comprehensive Identification of Bridge Genes to Explain the Progression from Chronic Hepatitis B Virus Infection to Hepatocellular Carcinoma.

BackgroundHepatitis B virus infection co-occurs in 33% of individuals with hepatocellular carcinoma worldwide. However, the molecular link between hepatitis B virus and hepatocellular carcinoma is unknown. Thus, we aimed to elucidate molecular linkages underlying pathogenesis through in-depth data mining analysis.Materials and MethodsDifferentially expressed genes were identified from patients with chronic hepatitis B virus infection, hepatocellular carcinoma, or both. Gene set enrichment analysis revealed signaling pathways involving differentially expressed genes. Protein-protein interaction networks, protein crosstalk, and enrichment were analyzed to determine whether differentially expressed gene products might serve as a bridge from hepatitis B virus infection to hepatocellular carcinoma pathogenesis. Prognostic potential and transcriptional and post-transcriptional regulators of bridge genes were also examined.ResultsWe identified vital bridge factors in hepatitis B virus infection-associated hepatocellular carcinoma. Differentially expressed genes were clustered into modules based on relative protein function. Signaling pathways associated with cancer, inflammation, immune system, and microenvironment showed significant crosstalk between modules. Thirty-two genes were dysregulated in hepatitis B virus infection-mediated hepatocellular carcinoma. CPEB3, RAB26, SLCO1B1, ST3GAL6 and XK had higher connectivity in the modular network, suggesting significant associations with survival. CDC20 and NUP107 were identified as driver genes as well as markers of poor prognosis.ConclusionOur results suggest that the sustained inflammatory environment created by hepatitis B virus infection is a risk factor for hepatocellular carcinoma. The identification of hepatitis B virus infection-related hepatocellular carcinoma bridge genes provides testable hypotheses about the pathogenesis of hepatocellular carcinoma.

ORMDL2 Deficiency Potentiates the ORMDL3-Dependent Changes in Mast Cell Signaling.

The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO exhibited increased intracellular levels of sphingosine-1-phosphate (S1P). Furthermore, we found that concurrent ORMDL2 and ORMDL3 deficiency increased IκB-α phosphorylation, degranulation, and production of IL-4, IL-6, and TNF-α cytokines in antigen-activated mast cells. Interestingly, the chemotaxis towards antigen was increased in all mutant cell types analyzed. Experiments in vivo showed that passive cutaneous anaphylaxis (PCA), which is initiated by mast cell activation, was increased only in ORMDL2,3 double KO mice, supporting our in vitro observations with mast cells. On the other hand, ORMDL3 KO and ORMDL2,3 double KO mice showed faster recovery from passive systemic anaphylaxis, which could be mediated by increased levels of blood S1P presented in such mice. Our findings demonstrate that Ormdl2 deficiency potentiates the ORMDL3-dependent changes in mast cell signaling.

Inflammatory stress in SARS-COV-2 associated Acute Kidney Injury

Increasing clinical evidence shows that acute kidney injury (AKI) is a common and severe complication in critically ill COVID-19 patients. The older age, the severity of COVID-19 infection, the ethnicity, and the history of smoking, diabetes, hypertension, and cardiovascular disease are the risk factor for AKI in COVID-19 patients. Of them, inflammation may be a key player in the pathogenesis of AKI in patients with COVID-19. It is highly possible that SARS-COV-2 infection may trigger the activation of multiple inflammatory pathways including angiotensin II, cytokine storm such as interleukin-6 (IL-6), C-reactive protein (CRP), TGF-β signaling, complement activation, and lung-kidney crosstalk to cause AKI. Thus, treatments by targeting these inflammatory molecules and pathways with a monoclonal antibody against IL-6 (Tocilizumab), C3 inhibitor AMY-101, anti-C5 antibody, anti-TGF-β OT-101, and the use of CRRT in critically ill patients may represent as novel and specific therapies for AKI in COVID-19 patients.