Proteasome activator 28γ (PA28γ) allosterically activates trypsin-like proteolysis by binding to the α-ring of the 20S proteasome

Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis. Here, by mechanistically decoupling gating effects from active site effects, we unambiguously demonstrate that WT PA28γ allosterically activates the T-L site. We show PA28γ binding increases the Kcat/Km by 13-fold for T-L peptide substrates while having little-to-no effect on hydrolysis kinetics for CT-L or C-L substrates. Furthermore, mutagenesis and domain swaps of PA28γ reveal that it does not select for T-L peptide substrates through either the substrate entry pore or the distal intrinsically disordered region. We also show that a previously reported point mutation can functionally switch PA28γ from a T-L activating to a gate-opening activator in a mutually exclusive fashion. Finally, using cryogenic electron microscopy, we visualized the PA28γ-proteasome complex at 4.3 Å and confirmed its expected quaternary structure. The results of this study provide unambiguous evidence that PA28γ can function by binding the 20S proteasome to allosterically activate the T-L proteolytic site.     

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

The integrin receptor α_Mβ₂ mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of α_Mβ₂ signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human α_Mβ₂ headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H α_M variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the α_M subunit in an Mg²⁺-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the α_M subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound α_Mβ₂ with iC3b, suggesting that it may represent a novel high-affinity proteinaceous α_Mβ₂-specific agonist. Taken together, our data suggest that the iC3b–α_Mβ₂ complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to α_Mβ₂.     

The GPCR–β-arrestin complex allosterically activates C-Raf by binding its amino terminus

G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes in vitro. Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.     

Finite element modeling of α-helices and tropocollagen molecules referring to spike of SARS-CoV-2

The newly developed finite element (FE) modeling at the atomic scale was used to predict the static and dynamic response of the α-helix (AH) and tropocollagen (TC) protein fragments, the main building blocks of the spike of the SARS-CoV-2. The geometry and morphology of the spike’s stalk and its connection to the viral envelope were determined from the combination of most recent molecular dynamics (MD) simulation and images of cryoelectron microscopy. The stiffness parameters of the covalent bonds in the main chain of the helix were taken from the literature. The AH and TC were modeled using both beam elements (wire model) and shell elements (ribbon model) in FE analysis to predict their mechanical properties under tension. The asymptotic stiffening features of AH and TC under tensile loading were revealed and compared with a new analytical solution. The mechanical stiffnesses under other loading conditions, including compression, torsion, and bending, were also predicted numerically and correlated with the results of the existing MD simulations and tests. The mode shapes and natural frequencies of the spike were predicted using the built FE model. The frequencies were shown to be within the safe range of 1–20 MHz routinely used for medical imaging and diagnosis by means of ultrasound. These results provide a solid theoretical basis for using ultrasound to study damaging coronavirus through transient and resonant vibration at large deformations.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this β7–β8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by β7–β8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.     

PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide– and natriuretic peptide–induced cGMP generation. However, some reports have shown that PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu²⁺ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGIα Cys⁴³, Cys¹¹⁸, and Cys¹⁹⁶ play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys⁴³ crosslinking in response to H₂O₂ (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.     

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN–CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2–binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against α_v and β₃ but not α₅ or β₁ integrins. Similarly, we observed direct binding of S1-RBD to recombinant human α_vβ₃ and α_vβ₆ integrins, but not α₅β₁ integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an α_v-selective integrin agonist. This study provides evidence that cell surface α_v-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Sensitive UHPLC-MS/MS quantification method for 4β- and 4α-hydroxycholesterol in plasma for accurate CYP3A phenotyping

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3–5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity.Supplementary key words: cholesterol, cytochrome P450, kidney, kinetics, pharmacokinetics, 4β-hydroxycholesterol, 4α-hydroxycholesterol, cytochrome P450 3A, mass spectrometry, plasma

Pharmacokinetics of drugs show large interindividual variability, and some drug-metabolizing enzymes and transporters are involved in the variability. Cytochrome P450 (CYP)3A is a major subfamily of metabolic enzymes involved in the metabolism of some drugs in the liver and small intestine (1). The main isoenzymes of this subfamily are CYP3A4 and CYP3A5. There is a large interindividual variability in CYP3A activity among patients, and the variability was reported to affect the clinical efficacy and the adverse reaction of CYP3A substrate drugs (2, 3). Thus, phenotyping of CYP3A activity is clinically important for more effective and safer treatment by CYP3A substrate drugs.Midazolam has been reported to be useful and considered a standard probe for CYP3A phenotyping (4, 5). Although midazolam is commonly used in drug-drug interaction studies (6, 7, 8, 9), this drug has some limitations in clinical application. For example, multiple blood samplings are needed to calculate the clearance for phenotyping, which limits its use in infants and elderly people. Midazolam shows high protein binding especially to albumin (approximately 96%) (10), and the free fraction may increase in patients with lower albumin levels, resulting in apparently increased hepatic clearance. Thus, phenotyping using midazolam may not be suitable in some patients with liver disease such as cirrhosis or kidney failure.To overcome these problems, 4β-hydroxycholesterol (4β-OHC) has drawn attention as an endogenous phenotyping probe for CYP3A activity. 4β-OHC is formed by CYP3A4 and CYP3A5 (11, 12) and has a long plasma half-life (approximately 17 days) (13). Since there is no circadian change in plasma 4β-OHC concentrations, one-point blood sampling is sufficient for CYP3A phenotyping. 4β-OHC is slowly metabolized by CYP7A1 (14), and CYP7A1 activity is not affected by kidney failure (15). Therefore, plasma 4β-OHC concentration is a suitable probe for CYP3A phenotyping in infants, elderly people, and patients with kidney failure or liver diseases including cirrhosis (16, 17, 18, 19, 20, 21).Several quantification methods have been reported for the measurement of plasma 4β-OHC concentrations using gas chromatography coupled to mass spectrometry (11) and high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (22, 23, 24, 25, 26). Recently, Hautajärvi et al. (27) reported an ultra-high performance liquid chromatography coupled to high resolution mass spectrometry method for quantification of plasma 4β-OHC and 4α-hydroxycholesterol (4α-OHC) concentrations. 4α-OHC, a stereoisomer of 4β-OHC, is formed via autooxidation of cholesterol, and not by CYP3A. Therefore, plasma 4α-OHC concentration reflects plasma sample stability, because plasma 4α-OHC concentration increases in uncontrolled storage condition (28). Furthermore, oxysterols including 4β-OHC and 4α-OHC have been reported to be elevated by cholesterol autoxidation due to oxidative stress or inflammation in the liver, regardless of CYP3A activity (29). Thus, simultaneous quantification of 4β-OHC and 4α-OHC is preferred for phenotyping of CYP3A activity using clinical plasma samples.In this study, we established a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was applied to measure plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers and patients with chronic kidney disease (CKD).     

VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.