SHP-1 is a cytosolic protein-tyrosine phosphatase that behaves as a negative regulator in eukaryotic cellular signaling pathways. To understand its regulatory mechanism, we have determined the crystal structure of the C-terminal truncated human SHP-1 in the inactive conformation at 2.8-Å resolution and refined the structure to a crystallographic R-factor of 24.0%. The three-dimensional structure shows that the ligand-free SHP-1 has an auto-inhibited conformation. Its N-SH2 domain blocks the catalytic domain and keeps the enzyme in the inactive conformation, which supports that the phosphatase activity of SHP-1 is primarily regulated by the N-SH2 domain. In addition, the C-SH2 domain of SHP-1 has a different orientation from and is more flexible than that of SHP-2, which enables us to propose an enzymatic activation mechanism in which the C-SH2 domains of SHPs could be involved in searching for phosphotyrosine activators.Tyrosine phosphorylation is a key mechanism for regulating eukaryotic cellular signaling pathways. The protein tyrosine phosphorylation level is precisely regulated by two types of enzymes: protein-tyrosine kinases (PTKs) 1 and protein-tyrosine phosphatases (PTPs), in which PTPs act to counter-balance the process through dephosphorylation of the phosphorylated tyrosines (1, 2). PTPs can be divided into two groups, receptor protein-tyrosine phosphatases and cytosolic proteintyrosine phosphatases. The SH2 domain-containing PTPs, SHP-1 and SHP-2, are both cytosolic PTPs and share many structural and regulatory features. They both have two tandem SH2 domains at the N terminus followed by a single catalytic domain and an inhibitory C-terminal tail. However, irrespective of similar structural and regulatory characteristics, these two enzymes have different biological function in vivo.Different from SHP-2, which is expressed in all kinds of tissues, SHP-1 is predominantly expressed in hematopoietic and epithelial cells and behaves mainly as a negative regulator of signaling pathways in lymphocytes (1, 2). SHP-1 is dormant in the cytosol, with its phosphatase activity inhibited by both the SH2 domains and the C-terminal tail (1,(3)(4)(5). In response to an activation signal, SHP-1 is recruited to membrane-bound inhibitory receptors via the binding of its SH2 domains to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibitory motif within the cytoplasmic domain of a receptor (6 -8). During this process, SHP-1 undergoes a structural rearrangement, exposes its active site, and binds to the downstream substrates, thereby dephosphorylating the substrates to turn off the cellular signals.SHP-1 also presents in several types of non-hematopoietic cells (9 -12). Overexpression of a catalytically inactive SHP-1 mutant in these cells strongly suppressed mitogen-activated pathways, reducing signal transduction and activation of transcription; these findings demonstrate that SHP-1 has a positive effect on mitogenic signaling in these non-hematopoietic cells (10, 11). Thus, SHP-1 probably has both the nega...

The crystal structures of the protein-tyrosine phosphatase SHP-1 catalytic domain and the complex it forms with the substrate analogue tungstate have been determined and refined to crystallographic R values of 0.209 at 2.5 Å resolution and 0.207 at 2.8 Å resolution, respectively. Despite low sequence similarity, the catalytic domain of SHP-1 shows high similarity in secondary and tertiary structures with other protein-tyrosine phosphatases (PTPs). In contrast to the conformational changes observed in the crystal structures of PTP1B and Yersinia PTP, the WPD loop (Trp 419 -Pro 428 ) in the catalytic domain of SHP-1 moves away from the substrate binding pocket after binding the tungstate ion. Sequence alignment and structural analysis suggest that the residues in the WPD loop, especially the amino acid following Asp 421 , are critical for the movement of WPD loop on binding substrates and the specific activity of protein-tyrosine phosphatases. Our mutagenesis and kinetic measurements have supported this hypothesis.Protein-tyrosine phosphatases (PTPs) 1 are a family of enzymes that catalyze the dephosphorylation of phosphotyrosine peptides. PTPs, together with the protein-tyrosine kinases, regulate the critical phosphotyrosine levels in the signal transduction pathways. PTPs can be divided into two groups: receptor-like PTPs and cytosolic PTPs. The receptor-like PTPs have highly conserved tandem intracellular catalytic domains and a diversity of receptor-like extracellular domains implicated in cell signaling. The cytosolic PTPs, however, contain a single conserved catalytic domain linked to a variety of noncatalytic segments that presumably exert a regulatory and/or targeting function. Among these noncatalytic segments are Src homology 2 (SH2) domains, which are found in SHPs, an extensively studied subfamily of intracellular PTPs. SHPs consist of SHP-1 (also called PTP1C, SH-PTP1, HCP, SHP, and PTPN6), SHP-2 (also called PTP2C, SH-PTP2, PTP1D, SH-PTP3, and Syp) and Csw from Drosophila (1-3). They contain two SH2 domains followed by a catalytic domain and an inhibitory C terminus. In biological systems, SHPs can be viewed as enzymes that exist primarily in the inactive state within resting cells. The inhibition of the activity of SHPs was attributable to the insertion of a DЈ-E loop (Asn 58 -Tyr 62 ) of the N-terminal SH2 domain into the substrate binding pocket (4). After cell stimulation, SHPs would translocate from cytosol to plasma membrane and bind to the tyrosine-phosphorylated receptors through their SH2 domains, becoming activated in the process. Although they belong to the same family and have similar catalytic and regulatory mechanisms, SHP-1 and SHP-2 have different biological functions in vivo.SHP-1 is highly expressed in hematopoietic cells. It has been identified as the gene responsible for causing the me and me v mouse phenotypes (5, 6), which cause profound abnormalities in the immune system. In addition, SHP-1 is one of the most extensively studied PTPs, functioning in hematopoietic cells as a t...

The recruitment of specific cytosolic proteins to intracellular membranes through binding phosphorylated derivatives of phosphatidylinositol (PtdIns) controls such processes as endocytosis, regulated exocytosis, cytoskeletal organization, and cell signaling. Protein modules such as FVYE domains and PH domains that bind specifically to PtdIns 3-phosphate (PtdIns-3-P) and polyphosphoinositides, respectively, can direct such membrane targeting. Here we show that two representative Phox homology (PX) domains selectively bind to specific phosphatidylinositol phosphates. The PX domain of Vam7p selectively binds PtdIns-3-P, while the PX domain of the CPK PI-3 kinase selectively binds PtdIns-4,5-P(2). In contrast, the PX domain of Vps5p displays no binding to any PtdInsPs that were tested. In addition, the double mutant (Y42A/L48Q) of the PX domain of Vam7p, reported to cause vacuolar trafficking defects in yeast, has a dramatically decreased level of binding to PtdIns-3-P. These data reveal that the membrane targeting function of the Vam7p PX domain is based on its ability to associate with PtdIns-3-P, analogous to the function of FYVE domains.

Chronic inflammation is the primary cause of gastric cancer (GC). NLRP3, as an important inflammasome component, has crucial roles in initiating inflammation. However, the potential roles of NLRP3 in GC is unknown. Here, we show that NLRP3 expression is markedly upregulated in GC, which promotes NLRP3 inflammasome activation and interleukin-1β (IL-1β) secretion in macrophages. In addition, NLRP3 binds to cyclin-D1 (CCND1) promoter and promotes its transcription in gastric epithelial cells. Consequently, NLRP3 enhances epithelial cells proliferation and GC tumorigenesis. Furthermore, we identify miR-22, which is constitutively expressed in gastric mucosa, as a suppressor of NLRP3. MiR-22 directly targets NLRP3 and attenuates its oncogenic effects in vitro and in vivo. However, Helicobacter pylori (H. pylori) infection suppresses miR-22 expression, while enhances NLRP3 expression, and that triggers uncontrolled proliferation of epithelial cells and the emergence of GC. Thus, our research describes a mechanism by which miR-22 suppresses NLRP3 and maintains homeostasis of gastric microenvironments and suggests miR-22 as a potential target for the intervention of GC.

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRP␣, a member of the signal-regulatory proteins. We show that the variable ␤5-loop-␤6 motif confers SHP-1 substrate specificity at the P-4 and further Nterminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in proteintyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1. Protein-tyrosine phosphatases (PTPs)1 consist of a diverse family of enzymes that play crucial roles in cell growth, differentiation, and transformation (1-3). They can be broadly divided into membrane-bound, receptor-like PTPs, and cytosolic PTPs. The cytosolic PTPs contain only one catalytic domain, whereas the membrane-bound receptor-like PTPs usually contain two tandem catalytic domains. The catalytic domains of PTPs are highly conserved in their three-dimensional structures (4 -7). However, they have remarkably different substrate specificity (3, 8 -10), which is still not well understood. Previous studies using various synthetic phosphotyrosyl peptides failed to identify a shared by PTP substrate because the peptides studied were not derived from physiological substrates of PTPs. In the present study, we have addressed the structural basis for the substrate specificity of PTPs using SHP-1 and its physiological substrate SIRP␣/SHPS-1 as a model. SIRP␣ is a transmembrane protein of the signal-regulatory protein family. Its extracellular domain contains three immunoglobulin domains, and its cytoplasmic domain contains four phosphotyrosine sites (Tyr(P) 427 , Tyr(P) 452 , Tyr(P) 469 , and Tyr(P) 495 ). SHP-1 is expressed primarily in hematopoietic cells, and contains two Src homology 2 (SH2) domains, a neighboring catalytic domain, and a C-terminal tail. Its phosphatase activity is inhibited by both the SH2 domains and the C-terminal tail (11,12). SHP-1 is activated upon the binding of its tandem SH2 domains to immunoreceptor tyrosine-based inhibitory motifs. Domain-swapping studies on SHP-1 and its analogue, SHP-2, have shown that the catalytic domains of SHP-1 and SHP-2 have distinct substrate specificity (9, 10), and therefore illustrate that the dissection of the structural basis for the substrate specificity of SHP-1 is fundamental to the understanding of its physiological functions. The identification of the substrates of SHP-1 (i.e. SIRP␣, CD22, and CD72; Refs. 13-15) has made it possible for us to probe this structural basis. The results of this probe are presented below. EXPERIMENTAL PROCEDURESCrystallization and Data Collection-The C455S mutant of the SHP-1 catalytic domain (245-532) was cloned, expressed, and purified as described elsewhere (16). The phosphotyrosyl decapeptides were synthesized and purified to ...

Covalent modifications of histone N-terminal tails play a critical role in regulating chromatin structure and controlling gene expression. These modifications are controlled by histone-modifying enzymes and read out by histone-binding proteins. Numerous proteins have been identified as histone modification readers. Here we report the family-wide characterization of histone binding abilities of human CW domain-containing proteins. We demonstrate that the CW domains in ZCWPW2 and MORC3/4 selectively recognize histone H3 trimethylated at Lys-4, similar to ZCWPW1 reported previously, while the MORC1/2 and LSD2 lack histone H3 Lys-4 binding ability. Our crystal structures of the CW domains of ZCWPW2 and MORC3 in complex with the histone H3 trimethylated at Lys-4 peptide reveal the molecular basis of this interaction. In each complex, two tryptophan residues in the CW domain form the "floor" and "right wall," respectively, of the methyllysine recognition cage. Our mutation results based on ZCWPW2 reveal that the right wall tryptophan residue is essential for binding, and the floor tryptophan residue enhances binding affinity. Our structural and mutational analysis highlights the conserved roles of the cage residues of CW domain across the histone methyllysine binders but also suggests why some CW domains lack histone binding ability.Chromatin structure is dynamically regulated by histone post-translational modifications, such as methylation, acetylation, phosphorylation, ubiquitination, and sumoylation (1). These post-translational modifications constitute the "histone code," which is written or erased by histone-modifying enzymes and recognized by histone code "reader" proteins (2-4).Histone methylation, such as lysine methylation at the ⑀-amino group at levels from mono-to trimethylation (me1-me3), has received extensive attention (5). A number of domains bind methylated histone tails. Prominent examples include the chromodomain, Tudor domain, MBT domain, PWWP domain, and PHD domain (4, 6, 7). The CW domain has recently been identified as a new member of the lysine methylation reader family (8 -11).The CW domain is a zinc binding domain, composed of ϳ50 amino acid residues with four conserved cysteine (C) and two conserved tryptophan (W) residues, and its name was derived from these conserved residues. CW domains are found in chromatin-associated proteins in animals and plants and grouped into 12 families based on sequence similarity (12). There are seven CW domain-containing proteins in humans, namely ZCWPW1, ZCWPW2, MORC1, MORC2, MORC3, MORC4, and LSD2 (Fig. 1A). Prior studies have shown that the CW domains of ZCWPW1 (8), MORC3 (10), and MORC4 (9) are readers of H3K4 3 methylated histones with differing preferences for histone H3K4 methylation states (i.e. ZCWPW1 and MORC3 preferentially recognize histone H3K4me3 (8, 10), whereas the CW domain of human MORC4 prefers dimethylated H3K4 (9)). The LSD2 CW domain is required for the demethylation function of LSD2 but does not bind to any H3K4 peptides (13). However, th...

Support vector machine (SVM)‐based multivariate pattern analysis (MVPA) has delivered promising performance in decoding specific task states based on functional magnetic resonance imaging (fMRI) of the human brain. Conventionally, the SVM‐MVPA requires careful feature selection/extraction according to expert knowledge. In this study, we propose a deep neural network (DNN) for directly decoding multiple brain task states from fMRI signals of the brain without any burden for feature handcrafts. We trained and tested the DNN classifier using task fMRI data from the Human Connectome Project's S1200 dataset (N = 1,034). In tests to verify its performance, the proposed classification method identified seven tasks with an average accuracy of 93.7%. We also showed the general applicability of the DNN for transfer learning to small datasets (N = 43), a situation encountered in typical neuroscience research. The proposed method achieved an average accuracy of 89.0 and 94.7% on a working memory task and a motor classification task, respectively, higher than the accuracy of 69.2 and 68.6% obtained by the SVM‐MVPA. A network visualization analysis showed that the DNN automatically detected features from areas of the brain related to each task. Without incurring the burden of handcrafting the features, the proposed deep decoding method can classify brain task states highly accurately, and is a powerful tool for fMRI researchers.

Recently, there has been significant progress in solving quantum many-particle problem via machine learning based on the restricted Boltzmann machine. However, it is still highly challenging to solve frustrated models via machine learning, which has not been demonstrated so far. In this work, we design a brand new convolutional neural network (CNN) to solve such quantum many-particle problems. We demonstrate, for the first time, of solving the highly frustrated spin-1/2 J1-J2 antiferromagnetic Heisenberg model on square lattices via CNN. The energy per site achieved by the CNN is even better than previous string-bond-state calculations. Our work therefore opens up a new routine to solve challenging frustrated quantum many-particle problems using machine learning.