Jin Zhang’s research while affiliated with China-Japan Friendship Hospital and other places

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Publications (414)


Differentiation of lung tissue-resident c-Kit+ cells into microvascular endothelial cells alleviates pulmonary vascular remodeling
  • Article

February 2025

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11 Reads

Developmental Cell

Jinqiu Li

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Ting Shu

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[...]

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Chen Wang



Biocircuitry linked to exercise adaptations: impact of dose and inter-individual response heterogeneity

January 2025

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31 Reads

In a randomized, dose-response trial, we used molecular and phenomic profiling to compare responses to traditional (TRAD) endurance plus resistance training and high-intensity tactical training (HITT). Ninety-four participants (18-27 years) completed 12 weeks of TRAD or HITT followed by 4 weeks of detraining. While in vivo phenotype improvements were not dose-dependent, a few dose-dependent ex vivo muscle adaptations were overshadowed by wide-ranging inter-individual response heterogeneity (IRH). To address this, we established minimum clinically important difference (MCID) scores to classify participants by their attainment of MCIDs for functional muscle quality (fMQ) and cardiorespiratory fitness (CRF). Using differential gene expression (DGE) of muscle and exosomal microRNAs (miRs) and higher-order singular value decomposition (HOSVD), we mapped the molecular and phenomic biocircuitry of IRH. Nine miRs emerged as robust features of training adaptability, providing new insights into the integrated biocircuitry driving exercise adaptations and response heterogeneity. In brief We examined in vivo and ex vivo adaptations to two randomized exercise prescriptions. Individual response variability overshadowed dose dependent effects. Deep phenotyping and miR transcriptomics of serum exosomes and skeletal muscle enabled multidimensional modeling of integrated biocircuits linked to attaining clinically significant outcomes.


Fig. 1 TIP60 and ΔNp63α are overexpressed in cisplatin-resistant cells. A A431 Parental and A431 Pt and B JHU029 and JHU006 cells were subjected to a 2-h cisplatin pulse treatment at the indicated doses. At 48-h post treatment, cell viability was measured by MTS assay (left panel). The y-axis indicates cell viability relative to vehicle-treated cells. The x-axis indicates the μg/μl concentration of cisplatin used for pulse treatment. Error bars represent ±1 SD from the mean. *p < 0.05 compared to respective control at each dose of cisplatin. Bar plots (right panel) show the mean IC 50 value calculated from three independent experiments. Error bars indicate the mean +1 SEM from three independent experiments. *p < 0.05 compared to the IC50 value of A431 Parental or JHU029 sensitive controls. Immunoblot analysis performed on C A431 Parental and A431 Pt D JHU029 and JHU006 cells using antibodies specific for p63 and TIP60. Fold change in ΔNp63α protein relative to respective control is listed above each band. β-actin was included as a loading control. Representative blots are shown.
Fig. 3 TIP60 protects ΔNp63α from cisplatin-mediated degradation. Immunoblot of A A431 Parental and A431 Pt cells treated either vehicle or cisplatin for 2-h pulse and harvested 24 h post pulse. B A431 Parental and A431 Pt cells were pulsed with cisplatin for 2 h and subsequently treated with cycloheximide 100 μg/ml cycloheximide (CHX) for 2, 4, 6, 8 and 10 h along with a non-treated control (0 h), respectively. The y-axis shows the percentage of ΔNp63α protein remaining relative to vehicle-treated cells at 0 h. ΔNp63α half-life (t ½ ) was determined from the exponential curve equation calculated using the one-phase exponential decay model in GraphPad Prism 6 (bottom panel). Error bars indicate mean ± SEM from three independent experiments. *p ≤ 0.05 relative to Parental. C Lenti A431-eGFP and Lenti A431-TIP60 stable cells treated with vehicle or cisplatin for 2-h pulse and harvested 24 h later. Immunoblot analysis was performed using antibodies specific for p63, TIP60 or β-actin. Fold change in ΔNp63α protein relative to respective vehicle-treated control is listed above each band in (A, C). β-actin was included as a loading control for equivalent protein.
Fig. 7 TIP60 and ΔNp63α inhibit cell-cycle arrest in cisplatin-resistant cells. A Percentage of cells progressing through G2/M at 24 and 48-h post 2-h cisplatin pulse treatment in A431 Parental and Pt cells measured by flow cytometry using FCS Express 4. The y-axis indicates % of cells in G2/M cell cycle phase. *p ≤ 0.05 compared to vehicle-treated condition for the corresponding cell line at 24 h post pulse. B A431 Parental and Pt cells were transfected with NSC, sip63 or siTIP60. Cells were pulsed with 12.5 μg/ml cisplatin for 2-h and subjected to flow analysis 48 h later. Cell cycle profiles were analyzed using Flow Cytometry. The y-axis indicates the percentage of cells in G2/M cell-cycle phase. *p ≤ 0.05 compared to Pt (NSC). Representative blot is shown. C A431 Pt cells were transfected with NSC, sip63 and siTIP60 and harvested at 48 h post transfection. D A431 Pt cells were treated with either Vehicle (DMSO) or 50 μM of TH1834, TIP60 specific inhibitor as indicated. At 24 h post treatment, cells were harvested, and total RNA was extracted, and p21 transcript levels were measured by TaqMan-based qRT-PCR. The y-axis indicates the fold change in transcript levels relative to the control. E A431 Pt and JHU006 cells were treated with either Vehicle (DMSO) or 50 μM of TH1834, TIP60 specific inhibitor as indicated. Changes in p21, ΔNp63α and TIP60 protein levels were measured by immunoblot analysis performed using antibodies specific for p21, p63, TIP60 or β-actin, as indicated. Fold change in ΔNp63α protein relative to respective NSC or vehicle-treated control is listed above each band in (B, C, E). β-actin was included as a loading control for equivalent protein. Densitometric analysis (top-panel) showing the fold change in p21 levels relative to Pt (Veh) and JHU006 (Veh) and normalized to β-actin. Error bars indicate means +1SEM from two (A) to three (B-E) independent experiments. *p ≤ 0.05 relative to Pt (NSC) as control.
Fig. 8 TIP60 and ΔNp63α inhibit cell death and apoptosis in cisplatin-resistant cells. A A431 Pt cells (left-panel) transfected with NSC, sip63 and siTIP60 and harvested at 48 h post transfection. A431 Pt cells (right-panel) were treated with either Vehicle (DMSO) or 50 μM of TH1834 as indicated. Cells were harvested, total RNA extracted, and PUMA transcript levels were measured by TaqMan-based qRT-PCR. The y-axis indicates the fold change in transcript levels relative to the corresponding NSC or vehicle (Veh) control. Error bars indicate means + 1SEM from three independent experiments. B A431 Parental and Pt cells were transfected with NSC, sip63 or siTIP60 and analyzed by flow cytometry at 48 h post transfection. The y-axis indicates the percentage of cells in Sub-G1 cell-cycle phase. Error bars indicate the mean + 1SEM from three independent experiments. Immunoblot analysis (bottom panel) was performed to measure the changes in ΔNp63α and TIP60 protein levels and confirm silencing. Immunoblot analysis was performed using antibodies specific for p63, TIP60 or β-actin. β-actin was included as a loading control for equivalent protein. Representative blot is shown. C A431 Parental and Pt cells were transfected with NSC, sip63 and siTIP60. Cells were pulsed with vehicle or 42 μg/ml of cisplatin for 2-h and were subjected to immunoblot analysis 48 h later. D A431 Parental and Pt cells pulse treated with vehicle or 42 μg/ml of cisplatin for 2-h followed by continuous treatment with either vehicle or TH1834 (50 μM) for 24 h. Immunoblot was performed using antibodies specific for γ-H2AX, PARP, cleaved PARP, cleaved caspase-3, p63, TIP60 or β-actin. Fold change in ΔNp63α protein relative to respective NSC or vehicle-treated control is listed above each band in (B, C, D). β-actin was included as a loading control for equivalent protein in each lane. Densitometric analysis (top panels) showing the fold change in cleaved-PARP and γ-H2AX levels normalized to β-actin. Error bars indicate as mean + 1SEM from three independent experiments. * p ≤ 0.05 relative to A vehicle-treated Pt cells and C Parental NSC (cisplatin-treated) D vehicle-treated Parental or Pt cells as control. Representative blot is shown.
TIP60 enhances cisplatin resistance via regulating ΔNp63α acetylation in SCC
  • Article
  • Full-text available

December 2024

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22 Reads

Cell Death and Disease

Non-melanoma skin cancer, including basal and squamous cell carcinoma, is the most common form of cancer worldwide, with approximately 5.4 million new cases diagnosed each year in the United States. While the chemotherapeutic drug cisplatin is often used to treat squamous cell carcinoma (SCC) patients, low response rates and disease recurrence are common. In this study, we show that TIP60 and ΔNp63α levels correlate with cisplatin resistance in SCC cell lines, suggesting that TIP60 contributes to the failure of platinum-based drugs in SCC by regulating the stability and transcriptional activity of ΔNp63α. Depletion of endogenous TIP60 or pharmacological inhibition of TIP60 led to a decrease in ΔNp63α protein and acetylation levels in multiple SCC cell lines. We showed that TIP60 upregulates ΔNp63α protein levels in cisplatin-resistant SCC cell lines by protecting it from cisplatin-mediated degradation and increasing its protein stability. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, while loss of either TIP60 or ΔNp63α induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Taken together, our study indicates that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance by promoting cell proliferation and inhibiting apoptosis. Furthermore, our data suggests that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers.

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Figure 1. Schematic of DAPD working principle. (a) DAPD substrate schematic. Laser projected on the substrate excites the fluorescent emission layer (R6G in PMMA). R6G emission light is coupled into SPP mode at the top gold surface. Specimen atop of the Au film is illuminated by the SPPs. The scattered light is detected by an objective and forms a dark field image. (b) Fourier space relationship between the SPP illumination light, free space k-vector (k 0 ), diffraction-limit detection range, and the actual information collected from the object. (c) Super-resolution DAPD image could be reconstructed by a pretrained CNN with a single-frame low-resolution PDF image as input.
Figure 3. Experimental demonstration of DAPD by using 200 nm polystyrene beads. (a, b, e, f) Low-resolution PDF image before neural network processing. (c, d, g, h) Super-resolution DAPD reconstructed images. (i) Cross-section profile of the object marked with a pair of arrows line in panels (a) and (c). (j) Cross-section profile of the object marked with a pair of arrows line in panels (e) and (g). Dark line represents the original low-resolution PDF image. Red circles represent the output image pixels. Red solid line represents the fitted curve of two Gaussian functions and the blue dashed lines display the single Gaussian curve from each bead. Scale bar = 1 μm.
Figure 4. Trained network performance on experiment data with cells. (a) PDF image of single COS-7 cell. Top-left is the diffraction-limit PDF image, and bottom-right is the network processed DAPD image. (b−d, h−j) Diffraction-limited PDF images acquired with a 40×/0.6NA objective. (e−g, k−m) DAPD super-resolved images. Scale bar = 2 μm in panel (a) and 1 μm in all other panels. All the cross-section profiles are magnified twice for better visualization.
Deep Learning Assisted Plasmonic Dark-Field Microscopy for Super-Resolution Label-Free Imaging

November 2024

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79 Reads

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1 Citation

Nano Letters

Dark-field microscopy (DFM) is a widely used imaging tool, due to its high-contrast capability in imaging label-free specimens. Traditional DFM requires optical alignment to block the oblique illumination, and the resolution is diffraction-limited to the wavelength scale. In this work, we present deep-learning assisted plasmonic dark-field microscopy (DAPD), which is a single-frame super-resolution method using plasmonic dark-field (PDF) microscopy and deep-learning assisted image reconstruction. Specifically, we fabricated a designed PDF substrate with surface plasmon polaritons (SPPs) illuminating specimens on the substrate. Dark field images formed by scattered light from the specimen are further processed by a pretrained convolutional neural network (CNN) using a simulation dataset based on the designed substrate and parameters of the detection optics. We demonstrated a resolution enhancement of 2.8 times on various label-free objects with a large potential for future improvement. We highlight our technique as a compact alternative to traditional DFM with a significantly enhanced spatial resolution.


Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals

November 2024

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67 Reads

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1 Citation

Chemical Reviews

Cellular function is controlled through intricate networks of signals, which lead to the myriad pathways governing cell fate. Fluorescent biosensors have enabled the study of these signaling pathways in living systems across temporal and spatial scales. Over the years there has been an explosion in the number of fluorescent biosensors, as they have become available for numerous targets, utilized across spectral space, and suited for various imaging techniques. To guide users through this extensive biosensor landscape, we discuss critical aspects of fluorescent proteins for consideration in biosensor development, smart tagging strategies, and the historical and recent biosensors of various types, grouped by target, and with a focus on the design and recent applications of these sensors in living systems.


Spatiotemporal orchestration of calcium-cAMP oscillations on AKAP/AC nanodomains is governed by an incoherent feedforward loop

October 2024

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66 Reads

The nanoscale organization of enzymes associated with the dynamics of second messengers is critical for ensuring compartmentation and localization of signaling molecules in cells. Specifically, the spatiotemporal orchestration of cAMP and Ca²⁺ oscillations is critical for many cellular functions. Previous experimental studies have shown that the formation of nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates the phase of Ca²⁺-cAMP oscillations from out-of-phase to in-phase. In this work, we develop computational models of the Ca²⁺/cAMP pathway and AKAP/AC nanodomain formation that give rise to the two important predictions: instead of an arbitrary phase difference, the out-of-phase Ca²⁺/cAMP oscillation reaches Ca²⁺ trough and cAMP peak simultaneously, which is defined as inversely out-of-phase; the in-phase and inversely out-of-phase oscillations associated with Ca²⁺-cAMP dynamics on and away from the nanodomains can be explained by an incoherent feedforward loop. Factors such as cellular surface-to-volume ratio, compartment size, and distance between nanodomains do not affect the existence of in-phase or inversely out-of-phase Ca²⁺/cAMP oscillation, but cellular surface-to-volume ratio and compartment size can affect the time delay for the inversely out-of-phase Ca²⁺/cAMP oscillation while the distance between two nanodomains does not. Finally, we predict that both the Turing pattern-generated nanodomains and experimentally measured nanodomains demonstrate the existence of in-phase and inversely out-of-phase Ca²⁺/cAMP oscillation when the AC8 is at a low level, consistent with the behavior of an incoherent feedforward loop. These findings unveil the key circuit motif that governs cAMP and Ca²⁺ oscillations and advance our understanding of how nanodomains can lead to spatial compartmentation of second messengers.


Functional inversion of circadian regulator REV-ERBα leads to tumorigenic gene reprogramming

October 2024

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82 Reads

Proceedings of the National Academy of Sciences

Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.


Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC

October 2024

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41 Reads

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2 Citations

Nature Chemical Biology

The protein kinase C (PKC) family of serine and threonine kinases, consisting of three distinctly regulated subfamilies, has been established as critical for various cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs, is unclear. Here we present a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2) that enables the detection of minute changes (equivalent to 0.2% of maximum stimulation) in subcellular PKC activity. Using ExRai-CKAR2 with an enhanced diacylglycerol (DAG) biosensor, we uncover that G-protein-coupled receptor stimulation triggers sustained PKC activity at the endoplasmic reticulum and lysosomes, differentially mediated by Ca²⁺-sensitive conventional PKC and DAG-sensitive novel PKC, respectively. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or partitioning defective complexes, further enabled us to detect previously inaccessible endogenous atypical PKC activity in three-dimensional organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.


Citations (53)


... In general, precisely which metric(s) are chosen for optimisation depends on the application in mind, be it utilising MFE or ODMR, or favouring magnitude or speed of response as was of interest for the lock-in and multiplexing applications we demonstrated. As such -much like fluorescent proteins -we expect MFPs will also be engineered to make general improvements, such as to solubility, photo-stability and quantum-yield [52]. Finally, we hope that the development of MFPs can serve as the starting point for magnetically controlled biological actuators, whereby application of a local magnetic field can have downstream cellular effectssuch a technology would be of significant biomedical and biotechnological interest. ...

Reference:

Quantum Correlations in Engineered Magneto-Sensitive Fluorescent Proteins Enables Multi-Modal Sensing in Living Cells
Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals

Chemical Reviews

... This novel approach uses circularly permuted FP as the reporting unit, resulting in phosphorylation-dependent ratiometric changes of emission upon excitation at two different wavelengths. ExRai sensors have demonstrated unprecedented sensitivity compared to FRET-based activity sensors for kinases such as PKA, PKC, AMPK, or Akt [16][17][18][19][20] . ...

Sensitive fluorescent biosensor reveals differential subcellular regulation of PKC

Nature Chemical Biology

... The idea of PKA sequestration and aggregation in mammalian cells has recently been approached by several investigators. [67][68][69] Our proposal is in line with a recently described non-canonical PKA activation mechanism via aggregation of the R1a subunit in inherited Carney complex mutations, which rendered it incapable of inhibiting the C subunit. 67 Studies from the Zhang laboratory have provided evidence that both the mammalian Ca and R1a subunits are co-recruited into R1a bodies that act as a means of compartmentalizing cAMP. ...

Molecular determinants and signaling effects of PKA RIα phase separation
  • Citing Article
  • March 2024

Molecular Cell

... This property has been recently exploited for the design of biosensors, based on genetically encoded sensing motifs fused to HaloTag in which an analyte-dependent conformational change alters the equilibrium and hence the fluorescence of the dye. [36][37][38][39] Here, we repurpose this approach to engineer a photoswitchable fluorescent system by integrating a light-responsive protein domain into the HaloTag protein ( Figure 1). Upon illumination, the conformational change of the protein photoswitch alters the dye environment, shifting its equilibrium toward the open, fluorescent state. ...

Far-red chemigenetic biosensors for multi-dimensional and super-resolved kinase activity imaging

... A diverse array of analytical techniques have been employed to quantify glucose concentrations, encompassing fluorescence [5], electrochemical methods [6], and chemiluminescence [7]. Among these techniques, electrochemical sensors stand out prominently owing to their exceptional sensitivity, remarkable selectivity, and low detection limit. ...

Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism
  • Citing Article
  • February 2024

Annual Review of Biophysics

... Fluorescent biosensors have been remarkably improved in recent years, making them indispensable tools for studying intracellular signal transduction due to their ability to monitor signaling dynamics in real time within living cells [12][13][14]. Fluorescent biosensor imaging techniques such as fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and translocation-based biosensors have been developed to analyze cellular signaling and behavior in live cells [15][16][17]. Although obtaining robust FRET and BiFC signals, these techniques require a tedious optimization procedure to determine the relative locations of fluorophores and binding pairs as well as appropriate linker domains. ...

Genetically Encodable Biosensors for Ras Activity

... We hypothesized that decreased AMP might be related to reduced generation of adenosine under hypoxic condition. And the decrease of adenosine and AMP may result in pulmonary vascular endothelial dysfunction and increase vascular resistance, prompting pulmonary hypertension (PH) 27 . It has been reported that perturbed purine metabolism is associated with HAPE and hypoxia-induced PH 11,27,28 . ...

Inactivation of Malic Enzyme 1 in Endothelial Cells Alleviates Pulmonary Hypertension

Circulation

... As a multifunctional enzyme with deacetylase, desuccinylase, defatty-acylase, debutyrylase, deglutarylase, decrotonylase, mono-adenosine 5 ′ -diphosphate (ADP)-ribosyltransferase, and an NAD + -independent RNA deacetylase, SIRT7 is involved in various cellular processes such as transcriptional regulation, cellular metabolism, cell proliferation, and the maintenance of genome stability [6]. In recent years, accumulating evidence has underscored the regulatory role of SIRT7 in angiogenesis in different contexts [7][8][9][10][11][12][13]. However, the substrate-mediated molecular mechanisms remain unclear and fragmented. ...

Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through KLF4 deacetylation

Cardiovascular Research

... Previous studies suggested that Rac1 could be a potential therapeutic target for HNSCC, as evident by its tumor-specific overexpression, its elevated expression in relapsed vs. primary HNSCC [45], and its demonstrated contribution to HNSCC chemo-radioresistance [45], cell invasion (via both ∆Np63α/Rac1 and EGFR/Vav2/Rac1 axes) [22,54], adhesion and migration (via the RAPI-Rac1 axis) [21], etc. Here, our demonstration that RAC1-amplified and RAC1-A159V-mutated HNSCC could display preferential sensitivity to Rac inhibitor targeting in vivo further provides supportive evidence for an expansion of efforts in both preclinical and clinical development of Rac inhibitors for the treatment of HNSCC, as well as other cancers driven by RAC1 aberrations. ...

Np63α inhibits Rac1 activation and cancer cell invasion through suppression of PREX1

Cell Death Discovery

... As alternatives to a classical second messenger role of nucleosides, physiological activation of the PKA holoenzyme in vivo may be co-activated by nucleoside binding together with a second trigger like a posttranslational modification, redox state, specific protein-protein interaction or kinase regulation by liquid-liquid phase separation (Lopez-Palacios and Andersen 2023 ; Hardy et al. 2023 ). These triggers may allosterically shift the affinity or may be required for the final activating conformational change upon binding (Khamina et al. 2022 ), giving nucleosides a more auxiliary role in the allosteric kinase regulation. ...

Molecular Determinants and Signaling Effects of PKA RIα Phase Separation
  • Citing Preprint
  • December 2023