Phase Separation of Disease-Associated SHP2 Mutants Underlies MAPK Hyperactivation.

The non-receptor protein tyrosine phosphatase (PTP) SHP2, encoded by PTPN11, plays an essential role in RAS-mitogen-activated protein kinase (MAPK) signaling during normal development. It has been perplexing as to why both enzymatically activating and inactivating mutations in PTPN11 result in human developmental disorders with overlapping clinical manifestations. Here, we uncover a common liquid-liquid phase separation (LLPS) behavior shared by these disease-associated SHP2 mutants. SHP2 LLPS is mediated by the conserved well-folded PTP domain through multivalent electrostatic interactions and regulated by an intrinsic autoinhibitory mechanism through conformational changes. SHP2 allosteric inhibitors can attenuate LLPS of SHP2 mutants, which boosts SHP2 PTP activity. Moreover, disease-associated SHP2 mutants can recruit and activate wild-type (WT) SHP2 in LLPS to promote MAPK activation. These results not only suggest that LLPS serves as a gain-of-function mechanism involved in the pathogenesis of SHP2-associated human diseases but also provide evidence that PTP may be regulated by LLPS that can be therapeutically targeted.

Leptin and insulin act on POMC neurons to promote the browning of white fat.

The primary task of white adipose tissue (WAT) is the storage of lipids. However, "beige" adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here, we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning.

PTPN9 dephosphorylates FGFR2 pY656/657 through interaction with ACAP1 and ameliorates pemigatinib effect in cholangiocarcinoma.

ABSTRACT AND AIM: Cholangiocarcinoma (CCA) is a highly aggressive and lethal cancer that originates from the biliary epithelium. Systemic treatment options for CCA are currently limited, and the first targeted drug of CCA, pemigatinib, emerged in 2020 for CCA treatment by inhibiting FGFR2 phosphorylation. However, the regulatory mechanism of FGFR2 phosphorylation is not fully elucidated. APPROACH AND RESULTS: Here we screened the FGFR2-interacting proteins and showed that protein tyrosine phosphatase (PTP) N9 interacts with FGFR2 and negatively regulates FGFR2 pY656/657 . Using phosphatase activity assays and modeling the FGFR2-PTPN9 complex structure, we identified FGFR2 pY656/657 as a substrate of PTPN9, and found that sec. 14p domain of PTPN9 interacts with FGFR2 through ACAP1 mediation. Coexpression of PTPN9 and ACAP1 indicates a favorable prognosis for CCA. In addition, we identified key amino acids and motifs involved in the sec. 14p-APCP1-FGFR2 interaction, including the "YRETRRKE" motif of sec. 14p, Y471 of PTPN9, as well as the PH and Arf-GAP domain of ACAP1. Moreover, we discovered that the FGFR2 I654V substitution can decrease PTPN9-FGFR2 interaction and thereby reduce the effectiveness of pemigatinib treatment. Using a series of in vitro and in vivo experiments including patient-derived xenografts (PDX), we showed that PTPN9 synergistically enhances pemigatinib effectiveness and suppresses CCA proliferation, migration, and invasion by inhibiting FGFR2 pY656/657 . CONCLUSIONS: Our study identifies PTPN9 as a negative regulator of FGFR2 phosphorylation and a synergistic factor for pemigatinib treatment. The molecular mechanism, oncogenic function, and clinical significance of the PTPN9-ACAP1-FGFR2 complex are revealed, providing more evidence for CCA precision treatment.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Valosin-Containing Protein (VCP)/p97 Oligomerization.

Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.

PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

Discovery of flavone-derivatives as the new skeleton of transient receptor potential vanilloid 3 channel antagonists.

Transient receptor potential vanilloid 3 (TRPV3) channel is a temperature-sensitive and Ca2+-permeable nonselective cation channel, which is abundantly expressed in skin keratinocyte and plays an important role in skin homeostasis and repair. However, only a few TRPV3 inhibitors were reported. Few selective and potent modulators of the TRPV3 channel have hindered the progress of the investigation and clinical application. TRPV3 channel research still faces challenges and requires the new inhibitors. Flavonoids are a kind of natural compounds with various biological and pharmacological activities including anti-inflammatory and anti allergic effects, which is associated with some physiological effects mediated by TRPV3 channel. Herein, our group designed and synthesized a range of flavone derivatives, and investigated their inhibitory properties on the human TRPV3 channel by electrophysiology technique. Then, we identified a new potent TRPV3 antagonist 2d with IC50 of 0.62 µM. It also showed good selectivity on TRPV1, TRPV4, TRPA1 and TRPM8.

Natural Product-Inspired Molecules for Covalent Inhibition of SHP2 Tyrosine Phosphatase.

Natural products have been playing indispensable roles in the development of lifesaving drug molecules. They are also valuable sources for covalent protein modifiers. However, they often are scarce in nature and have complex chemical structures, which are limiting their further biomedical development. Thus, natural product-inspired small molecules which still contain the essence of the parent natural products but are readily available and amenable for structural modification, are important and desirable in searching for lead compounds for various disease treatment. Inspired by the complex and diverse ent-kaurene diterpenoids with significant biological activities, we have created a synthetically accessible and focused covalent library by incorporating the bicyclo[3.2.1]octane α-methylene ketone, which is considered as the pharmacophore of ent-kaurene diterpenoids, as half of the structure, and replacing the other half with much less complex but more druglike scaffolds. From this library, we have identified and characterized selective covalent inhibitors of protein tyrosine phosphatase SHP2, an important anti-cancer therapeutic target. The success of this study demonstrated the importance of creating and evaluating natural product-inspired library as well as their application in targeting challenging disease targets.

Lattice dynamics and heat transport in zeolitic imidazolate framework glasses.

The glassy state of zeolitic imidazolate frameworks (ZIFs) has shown great potential for energy-related applications, including solid electrolytes. However, their thermal conductivity (κ), an essential parameter influencing thermal dissipation, remains largely unexplored. In this work, using a combination of experiments, atomistic simulations, and lattice dynamics calculations, we investigate κ and the underlying heat conduction mechanism in ZIF glasses with varying ratios of imidazolate (Im) to benzimidazolate (bIm) linkers. The substitution of bIm for Im tunes the node-linker couplings but exhibits only a minor impact on the average diffusivity of low-frequency lattice modes. On the other hand, the linker substitution induces significant volume expansion, which, in turn, suppresses the contributions from lattice vibrations to κ, leading to decreased total heat conduction. Furthermore, spatial localization of internal high-frequency linker vibrations is promoted upon substitution, reducing their mode diffusivities. This is ascribed to structural deformations of the bIm units in the glasses. Our work unveils the detailed influences of linker substitution on the dual heat conduction characteristics of ZIF glasses and guides the κ regulation of related hybrid materials in practical applications.

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors.

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen-deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain-mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.

Nano-DESI Mass Spectrometry Imaging of Proteoforms in Biological Tissues with High Spatial Resolution.

Mass spectrometry imaging (MSI) is a powerful tool for label-free mapping of the spatial distribution of proteins in biological tissues. We have previously demonstrated imaging of individual proteoforms in biological tissues using nanospray desorption electrospray ionization (nano-DESI), an ambient liquid extraction-based MSI technique. Nano-DESI MSI generates multiply charged protein ions, which is advantageous for their identification using top-down proteomics analysis. In this study, we demonstrate proteoform mapping in biological tissues with a spatial resolution down to 7 µm using nano-DESI MSI. A substantial decrease in protein signals observed in high-spatial-resolution MSI makes these experiments challenging. We have enhanced the sensitivity of nano-DESI MSI experiments by optimizing the design of the capillary-based probe and the thickness of the tissue section. In addition, we demonstrate that oversampling may be used to further improve spatial resolution at little or no expense to sensitivity. These developments represent a new step in MSI-based spatial proteomics, which complements targeted imaging modalities widely used for studying biological systems.

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates.

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrate that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses reveal key residues that govern the interaction between PTP-MEG2 and its substrate, a peptide containing the phosphorylated NSF-pY83 site, specify PTP-MEG2 substrate selectivity, and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface reveals that PTP-MEG2 controls the fusion pore opening through NSF independent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we uncover that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 sites. Further structural and biochemical analyses confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies thus provide mechanistic insights in complex exocytosis processes.

Laminarin Reduces Cholesterol Uptake and NPC1L1 Protein Expression in High-Fat Diet (HFD)-Fed Mice.

Aberrantly high dietary cholesterol intake and intestinal cholesterol uptake lead to dyslipidemia, one of the risk factors for cardiovascular diseases (CVDs). Based on previous studies, laminarin, a polysaccharide found in brown algae, has hypolipidemic activity, but its underlying mechanism has not been elucidated. In this study, we investigated the effect of laminarin on intestinal cholesterol uptake in vitro, as well as the lipid and morphological parameters in an in vivo model of high-fat diet (HFD)-fed mice, and addressed the question of whether Niemann-Pick C1-like 1 protein (NPC1L1), a key transporter mediating dietary cholesterol uptake, is involved in the mechanistic action of laminarin. In in vitro studies, BODIPY-cholesterol-labeled Caco-2 cells were examined using confocal microscopy and a fluorescence reader. The results demonstrated that laminarin inhibited cholesterol uptake into Caco-2 cells in a concentration-dependent manner (EC50 = 20.69 µM). In HFD-fed C57BL/6J mice, laminarin significantly reduced the serum levels of total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). It also decreased hepatic levels of TC, TG, and total bile acids (TBA) while promoting the excretion of fecal cholesterol. Furthermore, laminarin significantly reduced local villous damage in the jejunum of HFD mice. Mechanistic studies revealed that laminarin significantly downregulated NPC1L1 protein expression in the jejunum of HFD-fed mice. The siRNA-mediated knockdown of NPC1L1 attenuated the laminarin-mediated inhibition of cholesterol uptake in Caco-2 cells. This study suggests that laminarin significantly improves dyslipidemia in HFD-fed mice, likely by reducing cholesterol uptake through a mechanism that involves the downregulation of NPC1L1 expression.

Mechanism of PRL2 phosphatase-mediated PTEN degradation and tumorigenesis.

Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) levels are frequently found reduced in human cancers, but how PTEN is down-regulated is not fully understood. In addition, although a compelling connection exists between PRL (phosphatase of regenerating liver) 2 and cancer, how this phosphatase induces oncogenesis has been an enigma. Here, we discovered that PRL2 ablation inhibits PTEN heterozygosity-induced tumorigenesis. PRL2 deficiency elevates PTEN and attenuates AKT signaling, leading to decreased proliferation and increased apoptosis in tumors. We also found that high PRL2 expression is correlated with low PTEN level with reduced overall patient survival. Mechanistically, we identified PTEN as a putative PRL2 substrate and demonstrated that PRL2 down-regulates PTEN by dephosphorylating PTEN at Y336, thereby augmenting NEDD4-mediated PTEN ubiquitination and proteasomal degradation. Given the strong cancer susceptibility to subtle reductions in PTEN, the ability of PRL2 to down-regulate PTEN provides a biochemical basis for its oncogenic propensity. The results also suggest that pharmacological targeting of PRL2 could provide a novel therapeutic strategy to restore PTEN, thereby obliterating PTEN deficiency-induced malignancies.

Discovery of a SHP2 Degrader with In Vivo Anti-Tumor Activity.

Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we designed and synthesized a novel series of proteolysis targeting chimeras (PROTACs) using a SHP2 allosteric inhibitor as warhead, with the goal of achieving SHP2 degradation both inside the cell and in vivo. Among these molecules, compound P9 induces efficient degradation of SHP2 (DC50 = 35.2 ± 1.5 nM) in a concentration- and time-dependent manner. Mechanistic investigation illustrates that the P9-mediated SHP2 degradation requires the recruitment of the E3 ligase and is ubiquitination- and proteasome-dependent. P9 shows improved anti-tumor activity in a number of cancer cell lines over its parent allosteric inhibitor. Importantly, administration of P9 leads to a nearly complete tumor regression in a xenograft mouse model, as a result of robust SHP2 depletion and suppression of phospho-ERK1/2 in the tumor. Hence, P9 represents the first SHP2 PROTAC molecule with excellent in vivo efficacy. It is anticipated that P9 could serve not only as a new chemical tool to interrogate SHP2 biology but also as a starting point for the development of novel therapeutics targeting SHP2.

Small Molecule Degraders of Protein Tyrosine Phosphatase 1B and T-Cell Protein Tyrosine Phosphatase for Cancer Immunotherapy.

Protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TC-PTP) play non-redundant negative regulatory roles in T-cell activation, tumor antigen presentation, insulin and leptin signaling, and are potential targets for several therapeutic applications. Here, we report the development of a highly potent and selective small molecule degrader DU-14 for both PTP1B and TC-PTP. DU-14 mediated PTP1B and TC-PTP degradation requires both target protein(s) and VHL E3 ligase engagement and is also ubiquitination- and proteasome-dependent. DU-14 enhances IFN-γ induced JAK1/2-STAT1 pathway activation and promotes MHC-I expression in tumor cells. DU-14 also activates CD8+ T-cells and augments STAT1 and STAT5 phosphorylation. Importantly, DU-14 induces PTP1B and TC-PTP degradation in vivo and suppresses MC38 syngeneic tumor growth. The results indicate that DU-14, as the first PTP1B and TC-PTP dual degrader, merits further development for treating cancer and other indications.

An Integrated Proteomic Strategy to Identify SHP2 Substrates.

Protein phosphatases play an essential role in normal cell physiology and the development of diseases such as cancer. The innate challenges associated with studying protein phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly coordinated networks. Here, we introduce a novel strategy using substrate-trapping mutants coupled with quantitative proteomics methods to identify physiological substrates of Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. The technique integrates three parallel mass spectrometry-based proteomics experiments, including affinity isolation of substrate-trapping mutant complex using wild-type and SHP2 KO cells, in vivo global quantitative phosphoproteomics, and in vitro phosphatase reaction. We confidently identified 18 direct substrates of SHP2 in the epidermal growth factor receptor signaling pathways, including both known and novel SHP2 substrates. Docking protein 1 was further validated using biochemical assays as a novel SHP2 substrate, providing a mechanism for SHP2-mediated Ras activation. This advanced workflow improves the systemic identification of direct substrates of protein phosphatases, facilitating our understanding of the equally important roles of protein phosphatases in cellular signaling.

Mechanistic insights explain the transforming potential of the T507K substitution in the protein-tyrosine phosphatase SHP2.

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

Functional interrogation and therapeutic targeting of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) counteract the enzymatic activity of protein tyrosine kinases to modulate levels of both normal and disease-associated protein tyrosine phosphorylation. Aberrant activity of PTPs has been linked to the progression of many disease states, yet no PTP inhibitors are currently clinically available. PTPs are without a doubt a difficult drug target. Despite this, many selective, potent, and bioavailable PTP inhibitors have been described, suggesting PTPs should once again be looked at as viable therapeutic targets. Herein, we summarize recently discovered PTP inhibitors and their use in the functional interrogation of PTPs in disease states. In addition, an overview of the therapeutic targeting of PTPs is described using SHP2 as a representative target.

The design and synthesis of transient receptor potential vanilloid 3 inhibitors with novel skeleton.

Transient receptor potential vanilloid 3 (TRPV3) channel as a member of thermo TRPV subfamily is primarily expressed in the keratinocytes of the skin and sensory neurons, and plays critical roles in various physiological and pathological processes such as inflammation, pain sensation and skin disorders. However, TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Recently, we synthesized a series of cinnamate ester derivatives and evaluated their inhibitory activities on human TRPV3 channels expressed in HEK293 cells using whole-cell patch clamp recordings. And, we identified two potent TRPV3 antagonists 7c and 8c which IC50 values were 1.05 µM and 86 nM, respectively. It also showed good selectivity to other subfamily members of TRPV, such as TRPV1 and TRPV4.