Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both phosphoserine/threonine and phosphotyrosine, play vital roles in immune activation, brain function and cell-growth signalling. A family-wide structural library of human DUSPs was constructed based on experimental structure determination supplemented with homology modelling. The catalytic domain of each individual DUSP has characteristic features in the active site and in surface-charge distribution, indicating substrate-interaction specificity. The active-site loop-to-strand switch occurs in a subtype-specific manner, indicating that the switch process is necessary for characteristic substrate interactions in the corresponding DUSPs. A comprehensive analysis of the activity-inhibition profile and active-site geometry of DUSPs revealed a novel role of the active-pocket structure in the substrate specificity of DUSPs. A structure-based analysis of redox responses indicated that the additional cysteine residues are important for the protection of enzyme activity. The family-wide structures of DUSPs form a basis for the understanding of phosphorylation-mediated signal transduction and the development of therapeutics.

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Protein tyrosine phosphatase sigma (PTPσ) plays a vital role in neural development. The extracellular domain of PTPσ binds to various proteoglycans, which control the activity of 2 intracellular PTP domains (D1 and D2). To understand the regulatory mechanism of PTPσ, we carried out structural and biochemical analyses of PTPσ D1D2. In the crystal structure analysis of a mutant form of D1D2 of PTPσ, we unexpectedly found that the catalytic cysteine of D1 is oxidized to cysteine sulfenic acid, while that of D2 remained in its reduced form, suggesting that D1 is more sensitive to oxidation than D2. This finding contrasts previous observations on PTPα. The cysteine sulfenic acid of D1 was further confirmed by immunoblot and mass spectrometric analyses. The stabilization of the cysteine sulfenic acid in the active site of PTP suggests that the formation of cysteine sulfenic acid may function as a stable intermediate during the redox-regulation of PTPs.

Dual specificity protein phosphatase 4 (DUSP4) has been considered a promising target for the development of therapeutics for various human cancers. Here, we report the first example for a successful application of the structure-based virtual screening to identify the novel small-molecule DUSP4 inhibitors. As a consequence of the virtual screening with the modified scoring function to include an effective molecular solvation free energy term, five micromolar DUSP4 inhibitors are found with the associated IC 50 values ranging from 3.5 to 10.8 μM. Because these newly identified inhibitors were also screened for having desirable physicochemical properties as a drug candidate, they may serve as a starting point of the structure-activity relationship study to optimize the medical efficacy. Structural features relevant to the stabilization of the new inhibitors in the active site of DUSP4 are discussed in detail.

The tumor microenvironment (TME) is a complex system composed of many cell types and an extracellular matrix (ECM). During tumorigenesis, cancer cells constantly interact with cellular components, biochemical cues, and the ECM in the TME, all of which make the environment favorable for cancer growth. Emerging evidence has revealed the importance of substrate elasticity and biomechanical forces in tumor progression and metastasis. However, the mechanisms underlying the cell response to mechanical signals-such as extrinsic mechanical forces and forces generated within the TME-are still relatively unknown. Moreover, having a deeper understanding of the mechanisms by which cancer cells sense mechanical forces and transmit signals to the cytoplasm would substantially help develop effective strategies for cancer treatment. This review provides an overview of biomechanical forces in the TME and the intracellular signaling pathways activated by mechanical cues as well as highlights the role of mechanotransductive pathways through mechanosensors that detect the altering biomechanical forces in the TME. BMB Rep. www.bmbreports.org

Cryogenic‐electron microscopy (cryo‐EM) is the preferred method to determine 3D structures of proteins and to study diverse material systems that intrinsically have radiation or air sensitivity. Current cryo‐EM sample preparation methods provide limited control over the sample quality, which limits the efficiency and high throughput of 3D structure analysis. This is partly because it is difficult to control the thickness of the vitreous ice that embeds specimens, in the range of nanoscale, depending on the size and type of materials of interest. Thus, there is a need for fine regulation of the thickness of vitreous ice to deliver consistent high signal‐to‐noise ratios for low‐contrast biological specimens. Herein, an advanced silicon‐chip‐based device is developed which has a regular array of micropatterned holes with a graphene oxide (GO) window on freestanding silicon nitride (SixNy). Accurately regulated depths of micropatterned holes enable precise control of vitreous ice thickness. Furthermore, GO window with affinity for biomolecules can facilitate concentration of the sample molecules at a higher level. Incorporation of micropatterned chips with a GO window enhances cryo‐EM imaging for various nanoscale biological samples including human immunodeficiency viral particles, groEL tetradecamers, apoferritin octahedral, aldolase homotetramer complexes, and tau filaments, as well as inorganic materials.

Although a wealth of persuasive evidence was provided for the involvement of dual specificity phosphatase 1 (DUSP1) in cancers, depression, and hepatitis C virus replication, small-molecule inhibitors have been reported rarely. Because most competitive phosphatase inhibitors are disadvantageous in terms of pharmacological properties due to the possession of highly polar groups, we aim in this work to identify the allosteric DUSP1 inhibitors through the structure-based virtual screening. Using the modified proteinligand binding energy function involving an accurate molecular dehydration term, we identify six allosteric DUSP1 inhibitors that impair the enzymatic activity at low-micromolar level. Consistent with the kinetic analysis on the inhibitory mechanism, docking simulation results indicate that the newly discovered inhibitors would bind in the peripheral binding pocket of DUSP1 in preference to the active site. Besides the micromolar-level biochemical potency, the allosteric DUSP1 inhibitors found in this work may be meritorious over the competitive ones because of the lack of the reactive moiety to mimic the substrate phosphate group. Hence, they are anticipated to serve as a good starting point for the development of new therapeutics against cancers and neurological diseases.

Mesenchymal stromal cells (MSCs) have attracted scientific and medical interest due to their self-renewing properties, pluripotency, and paracrine function. However, one of the main limitations to the clinical application of MSCs is their loss of efficacy after transplantation in vivo. Various bioengineering technologies to provide stem cell niche-like conditions have the potential to overcome this limitation. Here, focusing on the stem cell niche microenvironment, studies to maximize the immunomodulatory potential of MSCs by controlling biomechanical stimuli, including shear stress, hydrostatic pressure, stretch, and biophysical cues, such as extracellular matrix mimetic substrates, are discussed. The application of biomechanical forces or biophysical cues to the stem cell microenvironment will be beneficial for enhancing the immunomodulatory function of MSCs during cultivation and overcoming the current limitations of MSC therapy.