Phosphatase Src Homology Research Articles

Identification of a Compound Inhibiting Both the Enzymatic and Nonenzymatic Functions of Indoleamine 2,3-Dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune escape. Besides being a metabolic enzyme that catalyzes the first step of tryptophan catabolism, it also acts as a signal-transducing protein, whose partnering with tyrosine phosphatase Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate (SHPs) and phosphatidylinositol-3-kinase (PI3K) regulatory subunit p85 promotes the establishment of a sustained immunosuppressive phenotype. While IDO1 inhibitors typically interfere with its enzymatic activity, we aimed to discover a more effective modulator capable of blocking not only the enzymatic but also the signaling-mediated functions of IDO1. By virtual screening, we identified the compound VS-15, which selectively binds the heme-free form of IDO1, inhibits its enzymatic activity, and reduces the IDO1-mediated signaling pathway by negatively interfering with its partnership with SHPs and PI3K regulatory subunit p85 as well as with the IDO1 anchoring to the early endosomes in tumor cells. Moreover, VS-15 counteracts the TGF-β-mediated immunosuppressive phenotype in dendritic cells and reduces the level of inhibition of T cell proliferation by suppressive monocytes isolated from patients affected by pancreatic cancer. Herein, we describe the discovery and characterization of a small molecule with an unprecedented mechanism of action, capable of inhibiting both the enzymatic and nonenzymatic activities of IDO1 by binding to its apo-form. These results pave the way for the development of next-generation IDO1 inhibitors with a unique competitive advantage over the currently available modulators, thereby opening therapeutic opportunities in cancer immunotherapy.

Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2

Targeted protein degradation is an emergent and rapidly evolving therapeutic strategy. In particular, biologics-based targeted degradation modalities (bioPROTACs) are relatively under explored compared to small molecules. Here, we investigate how target affinity, cellular localization, and valency of bioPROTACs impact efficacy of targeted degradation of the oncogenic phosphatase src-homology 2 containing protein tyrosine phosphatase-2 (SHP2). We identify bivalent recruitment of SHP2 by bioPROTACs as a broadly applicable strategy to improve potency. Moreover, we demonstrate that SHP2-targeted bioPROTACs can effectively counteract gain-of-function SHP2 mutants present in cancer, which are otherwise challenging to selectively target with small molecule constructs. Overall, this study demonstrates the utility of bioPROTACs for challenging targets, and further explicates design principles for therapeutic bioPROTACs.

Interaction of the Src Homology Phosphatase 2 (Shp2) and Vimentin Modulates Glomerular Podocyte Function

Interaction of the Src Homology Phosphatase 2 (Shp2) and Vimentin Modulates Glomerular Podocyte Function

Regulation of PPARγ2 Stability and Activity by SHP-1.

The protein tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) plays an important role in modulating glucose and lipid homeostasis. We previously suggested a potential role of SHP-1 in the regulation of peroxisome proliferator-activated receptor γ2 (PPARγ2) expression and activity but the mechanisms were unexplored. PPARγ2 is the master regulator of adipogenesis, but how its activity is regulated by tyrosine phosphorylation is largely unknown. Here, we found that SHP-1 binds to PPARγ2 primarily via its N-terminal SH2-domain. We confirmed the phosphorylation of PPARγ2 on tyrosine-residue 78 (Y78), which was reduced by SHP-1 invitro resulting in decreased PPARγ2 stability. Loss of SHP-1 led to elevated, agonist-induced expression of the classical PPARγ2 targets FABP4 and CD36, concomitant with increased lipid content in cells expressing PPARγ2, an effect blunted by abrogation of PPARγ2 phosphorylation. Collectively, we discovered that SHP-1 affects the stability of PPARγ2 through dephosphorylation thereby influencing adipogenesis.

Tyrosine phosphatase SHP2 aggravates tumor progression and glycolysis by dephosphorylating PKM2 in gastric cancer.

Gastric cancer (GC) is among the most lethal human malignancies, yet it remains hampered by challenges in fronter of molecular-guided targeted therapy to direct clinical treatment strategies. The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) is involved in the malignant progression of GC. However, the detailed mechanisms of the posttranslational modifications of SHP2 remain poorly understood. Herein, we demonstrated that an allosteric SHP2 inhibitor, SHP099, was able to block tumor proliferation and migration of GC by dephosphorylating the pyruvate kinase M2 type (PKM2) protein. Mechanistically, we found that PKM2 is a bona fide target of SHP2. The dephosphorylation and activation of PKM2 by SHP2 are necessary to exacerbate tumor progression and GC glycolysis. Moreover, we demonstrated a strong correlation between the phosphorylation level of PKM2 and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in GC cells. Notably, the low phosphorylation expression of AMPK was negatively correlated with activated SHP2. Besides, we proved that cisplatin could activate SHP2 and SHP099 increased sensitivity to cisplatin in GC. Taken together, our results provide evidence that the SHP2/PKM2/AMPK axis exerts a key role in GC progression and glycolysis and could be a viable therapeutic approach for the therapy of GC.

Targeting Src homology phosphatase 2 ameliorates mouse diabetic nephropathy by attenuating ERK/NF-κB pathway-mediated renal inflammation

Renal inflammation is a pivotal mechanism underlying the pathophysiology of diabetic nephropathy (DN). The Src homology phosphatase 2 (SHP2) has been demonstrated to be linked to diabetes-induced inflammation, yet its roles and explicit molecular mechanisms in DN remain unexplored. Here, we report that SHP2 activity is upregulated in both DN patients and db/db mice. In addition, pharmacological inhibition of SHP2 with its specific inhibitor PHPS1 alleviates DN in db/db mice and attenuates renal inflammation. In vitro, PHPS1 administration prevents inflammatory responses in HK-2 cells stimulated by high glucose (HG). Mechanistically, PHPS1 represses HG-induced activation of the proinflammatory ERK/NF-κB signaling pathway, and these inhibitory effects are blocked in the presence of an ERK specific inhibitor, hence demonstrating that PHPS1 suppresses ERK/NF-κB pathway-mediated inflammation. Moreover, PHPS1 retards ERK/NF-κB pathway activation in db/db mice, and histologically, SHP2 activity is positively correlated with ERK/NF-κB activation in DN patients. Taken together, these findings identify SHP2 as a potential therapeutic target and show that its pharmacological inhibition might be a promising strategy to mitigate DN.-rV21qwpHSeiT1S94nwGvkVideo

Identification of linderalactone as a natural inhibitor of SHP2 to ameliorate CCl4-induced liver fibrosis.

Liver fibrosis is characterised by the activation of hepatic stellate cells (HSCs) and matrix deposition. Accumulating evidence has revealed that the oncogenic protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) acts as a therapeutic target of fibrosis. Although several SHP2 inhibitors have reached early clinical trials, there are currently no FDA-approved drugs that target SHP2. In this study, we aimed to identify novel SHP2 inhibitors from an in-house natural product library to treat liver fibrosis. Out of the screened 800 compounds, a furanogermacrane sesquiterpene, linderalactone (LIN), significantly inhibited SHP2 dephosphorylation activity in vitro. Cross-validated enzymatic assays, bio-layer interferometry (BLI) assays, and site-directed mutagenesis were used to confirm that LIN directly binds to the catalytic PTP domain of SHP2. In vivo administration of LIN significantly ameliorated carbon tetrachloride (CCl4)-induced HSC activation and liver fibrosis by inhibiting the TGFβ/Smad3 pathway. Thus, LIN or its derivatives could be considered potential therapeutic agents against SHP2-related diseases, such as liver fibrosis or NASH.

Long non-coding RNA FAM239A promotes tumor cell proliferation and migration by regulating tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 in head and neck squamous cell carcinoma

ObjectiveHead and neck squamous cell carcinoma (HNSCC), is one of the malignant tumors with high recurrence and metastasis. The family with sequence similarity (FAM) of non-coding RNAs promoted tumorigenesis and metastasis. But so far, long non-coding RNA (lncRNA) FAM239A’s function in HNSCC regulation remains unclear. This study aimed to explore the lncRNA FAM239A function and regulation mechanism in HNSCC cell proliferation and migration. DesignThe expression level of lncRNA FAM239A and tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) in HNSCC tumor tissue was tested by quantitative polymerase chain reaction. The cell proliferation and migration were tested by cell counting kit 8, kinetic live cell assay, and wound healing assay. The differential expression of SHP2 and immune infiltration in HNSCC were analyzed in the tumor immune estimation response and human protein atlas databases. And the survival analysis of SHP2 in HNSCC was analyzed in the gene expression profiling interactive analysis 2 databases. The SHP2 expression was tested by western blotting when lncRNA FAM239A overexpression and knockdown. ResultsLncRNA FAM239A and SHP2 were ectopically expressed in HNSCC tumor tissue. Cell proliferation and wound healing assays showed that lncRNA FAM239A promoted tumor cell proliferation and migration. SHP2 was overexpressed in HNSCC tumor tissue by database analyses, and the higher SHP2 expression caused poorer overall survival and disease-free survival in HNSCC patients. SHP2 expression was positively regulated by lncRNA FAM239A. ConclusionsLncRNA FAM239A promoted HNSCC cell proliferation and migration through upregulating SHP2 expression, which potentially provided new regulators for HNSCC.

Deficiency of the Src homology phosphatase 2 in podocytes is associated with renoprotective effects in mice under hyperglycemia.

Diabetic nephropathy (DN) is a significant complication of diabetes and the leading cause of end-stage renal disease. Hyperglycemia-induced dysfunction of the glomerular podocytes is a major contributor to the deterioration of renal function in DN. Previously, we demonstrated that podocyte-specific disruption of the Src homology phosphatase 2 (Shp2) ameliorated lipopolysaccharide-induced renal injury. This study aims to evaluate the contribution of Shp2 to podocyte function under hyperglycemia and explore the molecular underpinnings. We report elevated Shp2 in the E11 podocyte cell line under high glucose and the kidney under streptozotocin- and high-fat diet-induced hyperglycemia. Consistently, Shp2 disruption in podocytes was associated with partial renoprotective effects under hyperglycemia, as evidenced by the preserved renal function. At the molecular level, Shp2 deficiency was associated with altered renal insulin signaling and diminished hyperglycemia-induced renal endoplasmic reticulum stress, inflammation, and fibrosis. Additionally, Shp2 knockdown in E11 podocytes mimicked the in vivo deficiency of this phosphatase and ameliorated the deleterious impact of high glucose, whereas Shp2 reconstitution reversed these effects. Moreover, Shp2 deficiency attenuated high glucose-induced E11 podocyte migration. Further, we identified the protein tyrosine kinase FYN as a putative mediator of Shp2 signaling in podocytes under high glucose. Collectively, these findings suggest that Shp2 inactivation may afford protection to podocytes under hyperglycemia and highlight this phosphatase as a potential target to ameliorate glomerular dysfunction in DN.

Shp2 suppresses fat accumulation in white adipose tissue by activating Wnt/β-catenin signaling following vertical sleeve gastrectomy in obese rats with type-2 diabetes.

Adipogenesis and fat accumulation are closely associated with the development of obesity. Sleeve gastrectomy (SG) is an effective treatment for obesity and associated metabolic disorders. Leptin is downregulated after SG and Src homology phosphatase 2 (Shp2) has an important role in leptin signaling. The role of Shp2 in SG and the mechanisms of fat reduction following SG were further investigated in the current study. Sham and SG operations were performed on obese type-2 diabetes model Sprague-Dawley rats. Primary pre-adipocytes were isolated from the inguinal white adipose tissue (ingWAT) of the rats. Shp2 expression in ingWAT pre-adipocytes was silenced using small interfering RNA transfection. Shp2 function was inhibited using the specific inhibitor, SHP099. In addition, Shp2 was overexpressed using lentivirus. Gene and protein expression analysis was performed after adipocyte differentiation. Furthermore, Shp2-overexpressing ingWAT pre-adipocytes treated with the β-catenin inhibitor, PNU-74654, were also used for gene and protein expression analysis. Adipogenic markers, including triglycerides, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (Cebpα), adiponectin, fatty acid-binding protein 4 and leptin, were examined. Compared with the sham, triglyceride, leptin, PPARγ and Cebpα levels were significantly reduced in the ingWAT from the SG group. Shp2 expression levels were reduced following leptin treatment. Moreover, genetic analysis demonstrated depot-specific adipogenesis following Shp2 silencing or inhibition in ingWAT pre-adipocytes. Conversely, Shp2 overexpression decreased the expression of adipogenic markers by enhancing β-catenin expression. PNU-74654 treatment abolished the downregulation of adipogenic markers caused by Shp2 overexpression. SG decreased leptin levels in ingWAT, which in turn upregulated Shp2, and Shp2 suppressed fat accumulation and adipogenic differentiation by activating the Wnt/β-catenin signaling pathway. Overall, this may represent a potential mechanism of fat reduction in SG, and Shp2 may serve as a potential therapeutic target for the treatment of obesity and type-2 diabetes.

TRPV1 Activation by Capsaicin Mediates Glucose Oxidation and ATP Production Independent of Insulin Signalling in Mouse Skeletal Muscle Cells.

Background: Insulin resistance (IR), a key characteristic of type 2 diabetes (T2DM), is manifested by decreased insulin-stimulated glucose transport in target tissues. Emerging research has highlighted transient receptor potential cation channel subfamily V member (TRPV1) activation by capsaicin as a potential therapeutic target for these conditions. However, there are limited data on the effects of capsaicin on cell signalling molecules involved in glucose uptake. Methods: C2C12 cells were cultured and differentiated to acquire the myotube phenotype. The activation status of signalling molecules involved in glucose metabolism, including 5’ adenosine monophosphate-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinase 2 (CAMKK2), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (AKT), and src homology phosphatase 2 (SHP2), was examined. Finally, activation of CAMKK2 and AMPK, and glucose oxidation and ATP levels were measured in capsaicin-treated cells in the presence or absence of TRPV1 antagonist (SB-452533). Results: Capsaicin activated cell signalling molecules including CAMKK2 and AMPK leading to increased glucose oxidation and ATP generation independent of insulin in the differentiated C2C12 cells. Pharmacological inhibition of TRPV1 diminished the activation of CAMKK2 and AMPK as well as glucose oxidation and ATP production. Moreover, we observed an inhibitory effect of capsaicin in the phosphorylation of ERK1/2 in the mouse myotubes. Conclusion: Our data show that capsaicin-mediated stimulation of TRPV1 in differentiated C2C12 cells leads to activation of CAMKK2 and AMPK, and increased glucose oxidation which is concomitant with an elevation in intracellular ATP level. Further studies of the effect of TRPV1 channel activation by capsaicin on glucose metabolism could provide novel therapeutic utility for the management of IR and T2DM.

Associations of INPPL1 (+1893CC/AA and + 2945AA/GG) exonic polymorphisms with the risk of type 2 diabetes mellitus in North Indian population: A case control study

Background and aimINPPL1 gene encodes a lipid phosphatase Src homology 2-containing 5′-inositol phosphatase 2 (SHIP2) which is involved in the negative regulation of insulin signaling. In the light of several association studies of INPPL1 gene polymorphisms with T2DM, and the absence of association studies from India, present study was conducted to assess the association of two SNPs of INPPL1 gene with T2DM risk in North Indian population. Material and methodTwo single nucleotide polymorphism (SNPs) (+1893CC/AA and + 2945AA/GG) in INPPL1 gene were genotyped in total of 270 subjects with PCR-RFLP genotyping method. ResultsIt was observed that, out of the two SNPs, only +1893CC/AA was found to be significantly associated with T2DM and frequency of A allele (C vs A, p = 0.002) has been found to be significantly higher in T2DM cases. Strong linkage disequilibrium was observed between two SNPs as assessed through D′ and r2 (D′ = 0.984, r2 = 0.009) and AG haplotype (OR = 3.59, 95% CI (1.59–9.80), p = 0.0015) was associated with increased risk of T2DM while CG haplotype (OR = 0.54, 95% CI (0.33–0.88) p = 0.012) significantly decreases the risk of developing T2DM. Regression analysis showed that SNP +1893CC/AA is associated with T2DM risk when adjusted for clinical/demographic variables. ConclusionResults showed that INPPL1 gene polymorphism +1893CC/AA may increase susceptibility to T2DM and ‘A' allele might be serving as a risk factor in development of T2DM in Indian population.

Conditional deletion of SRC-homology 2-containing protein tyrosine phosphatase 2 attenuates the severity of experimental osteoarthritis

Purpose: To investigate the roles of the tyrosine phosphatase Src-homology 2-containing protein tyrosine phosphatase 2 (SHP2) in macrophage polarization during the process of osteoarthritis (OA) induced by destabilization of the medial meniscus (DMM) surgery in Mice.

A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC50 = 0.32 μmol/L; WS-635: IC50 = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC50 = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.

Preservation of microvascular barrier function requires CD31 receptor-induced metabolic reprogramming

Endothelial barrier (EB) breaching is a frequent event during inflammation, and it is followed by the rapid recovery of microvascular integrity. The molecular mechanisms of EB recovery are poorly understood. Triggering of MHC molecules by migrating T-cells is a minimal signal capable of inducing endothelial contraction and transient microvascular leakage. Using this model, we show that EB recovery requires a CD31 receptor-induced, robust glycolytic response sustaining junction re-annealing. Mechanistically, this response involves src-homology phosphatase activation leading to Akt-mediated nuclear exclusion of FoxO1 and concomitant β-catenin translocation to the nucleus, collectively leading to cMyc transcription. CD31 signals also sustain mitochondrial respiration, however this pathway does not contribute to junction remodeling. We further show that pathologic microvascular leakage in CD31-deficient mice can be corrected by enhancing the glycolytic flux via pharmacological Akt or AMPK activation, thus providing a molecular platform for the therapeutic control of EB response.

Extracellular adenosine enhances pulmonary artery vasa vasorum endothelial cell barrier function via Gi/ELMO1/Rac1/PKA-dependent signaling mechanisms.

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.

Decrease in SHP-1 enhances myometrium remodeling via FAK activation leading to labor.

Preterm birth is one of the most common complications during human pregnancy and is associated with a dramatic switch within the uterus from quiescence to contractility. However, the mechanisms underlying uterine remodeling are largely unknown. Protein kinases and phosphatases play critical roles in regulating the phosphorylation of proteins involved in the smooth muscle cell functions. In the present study, we found that Src-homology phosphatase type-1 (SHP-1, PTPN6) was significantly decreased in human myometrium in labor compared with that not in labor. Timed-pregnant mice injected intraperitoneally with the specific SHP-1 inhibitor protein tyrosine phosphatase inhibitor I (PTPI-1) manifested significantly preterm labor, with enriched plasmalemmal dense plaques between myometrial cells and increased phosphorylation at Tyr397 and Tyr576/577 sites of focal adhesion kinase (FAK) in myometrial cells, which remained to the time of labor, whereas the phosphorylation levels of ERK1/2 and phosphatidylinositol 3 kinase (PI3K) showed a rapid increase upon PTPI-1 injection but fell back to normal at the time of labor. The Tyr576/577 in FAK played an important role in the interaction between FAK and SHP-1. Knockdown of SHP-1 dramatically increased the spontaneous contraction of human uterine smooth muscle cells (HUSMCs), which was reversed by coinfection of a FAK-knockdown lentivirus. PGF2α downregulated SHP-1 via PLCβ-PKC-NF-κB or PI3K-NF-κB pathways, suggesting the regenerative downregulation of SHP-1 enhances the uterine remodeling and plasticity by activating FAK and subsequent focal adhesion pathway, which eventually facilitates myometrium contraction and leads to labor. The study sheds new light on understanding of mechanisms that underlie the initiation of labor, and interventions for modulation of SHP-1 may provide a potential strategy for preventing preterm birth.

A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2)

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

Siglec-G Deficiency Ameliorates Hyper-Inflammation and Immune Collapse in Sepsis via Regulating Src Activation.

Hyper-inflammation during acute phase and sequential hypo-inflammation during immunosuppressive phase in macrophages/monocytes lead to multiorgan failure syndrome and immune collapse of sepsis, in which toll-like receptor (TLR)-triggered inflammatory responses play a major role. Here, we reported that Siglecg deficiency attenuated TLR4-triggered pro-inflammatory cytokine production and increased anti-inflammatory cytokine [interleukin-10 [IL-10]] production in vivo and in vitro at both acute and immunosuppressive phases. Siglecg deficiency also protected mice from lipopolysaccharide (LPS)-induced sepsis with less inflammation in the lung and less tissue destruction in the spleen. Siglec-G inhibited proto-oncogene tyrosine-protein kinase Src (Src) activation via recruiting and activating tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP1) through immunoreceptor tyrosine-based inhibitory motif (ITIM) domain. Src could inhibit TLR4-induced inflammatory cytokines and promote anti-inflammatory cytokine IL-10. Mechanical investigation showed that Src could interact with and phosphorylate STAT3. Src could also promote HIF1α degradation through activating GSK3β. Our study reveals that Siglec-G orchestrates TLR-induced inflammation, which outlines that blocking Siglec-G or activating Src may be a promising strategy for both acute and chronic inflammatory diseases.

STAT3: A Promising Therapeutic Target in Multiple Myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy for which novel treatment options are required. Signal Transducer and Activator of Transcription 3 (STAT3) overexpression in MM appears to be mediated by a variety of factors including interleukin-6 signaling and downregulation of Src homology phosphatase-1 (SHP-1). STAT3 overexpression in MM is associated with an adverse prognosis and may play a role in microenvironment-dependent treatment resistance. In addition to its pro-proliferative role, STAT3 upregulates anti-apoptotic proteins and leads to microRNA dysregulation in MM. Phosphatase of regenerating liver 3 (PRL-3) is an oncogenic phosphatase which is upregulated by STAT3. PRL-3 itself promotes STAT-3 phosphorylation resulting in a positive feedback loop. PRL-3 is overexpressed in a subset of MM patients and may cooperate with STAT3 to promote survival of MM cells. Indirectly targeting STAT3 via JAK (janus associated kinase) inhibition has shown promise in early clinical trials. Specific inhibitors of STAT3 showed in vitro efficacy but have failed in clinical trials while several STAT3 inhibitors derived from herbs have been shown to induce apoptosis of MM cells in vitro. Optimising the pharmacokinetic profiles of novel STAT3 inhibitors and identifying how best to combine these agents with existing anti-myeloma therapy are key questions to be addressed in future clinical trials.