Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.

"Sticki-ER": Functions of the Platelet Endoplasmic Reticulum.

Significance: The primary role of platelets is to generate a thrombus by platelet activation. Platelet activation relies on calcium mobilization from the endoplasmic reticulum (ER). ER resident proteins, which are externalized upon platelet activation, are essential for the function of platelet surface receptors and intercellular interactions. Recent Advances: The platelet ER is a conduit for changes in cellular function in response to the extracellular milieu. ER homeostasis is maintained by an appropriate redox balance, regulated calcium stores and normal protein folding. Alterations in ER function and ER stress results in ER proteins externalizing to the cell surface, including members of the protein disulfide isomerase family (PDIs) and chaperones. Critical Issues: The platelet ER is central to platelet function, but our understanding of its regulation is incomplete. Previous studies have focused on the function of PDIs in the extracellular space, and much less on their intracellular role. How platelets maintain ER homeostasis and how they direct ER chaperone proteins to facilitate intercellular signalling is unknown. Future Directions: An understanding of ER functions in the platelet is essential as these may determine critical platelet activities such as secretion and adhesion. Studies are necessary to understand the redox reactions of PDIs in the intracellular versus extracellular space, as these differentially affect platelet function. An unresolved question is how platelet ER proteins control calcium release. Regulation of protein folding in the platelet and downstream pathways of ER stress require further evaluation. Targeting the platelet ER may have therapeutic application in metabolic and neoplastic disease.

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Super-resolution imaging of linearized chromatin in tunable nanochannels.

Nanofluidic linearization and optical mapping of naked DNA have been reported in the research literature, and implemented in commercial instruments. However, the resolution with which DNA features can be resolved is still inherently limited by both Brownian motion and diffraction-limited optics. Direct analysis of native chromatin is further hampered by difficulty in electrophoretic manipulation, which is routinely used for DNA analysis. This paper describes the development of a three-layer, tunable, nanochannel system that enables non-electrophoretic linearization and immobilization of native chromatin. Furthermore, through careful selection of self-blinking fluorescent dyes and the design of the nanochannel system, we achieve direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging of the linearized chromatin. As an initial demonstration, rDNA chromatin extracted from Tetrahymena is analyzed by multi-color imaging of total DNA, newly synthesized DNA, and newly synthesized histone H3. Our analysis reveals a relatively even distribution of newly synthesized H3 across two halves of the rDNA chromatin with palindromic symmetry, supporting dispersive nucleosome segregation. As a proof-of-concept study, our work achieves super-resolution imaging of native chromatin fibers linearized and immobilized in tunable nanochannels. It opens up a new avenue for collecting long-range and high-resolution epigenetic information as well as genetic information.

Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow.

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation.

BACKGROUND: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines. OBJECTIVES: We hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI. METHODS: Biochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity. RESULTS: PDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis. CONCLUSION: PDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding.

BACKGROUND: The von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. OBJECTIVE: To determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF's platelet binding function. METHODS: We combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. RESULTS: We show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. CONCLUSIONS: Our data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.

Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model.

Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46, and ERp5, are required for in vivo thrombus formation in mice. Platelets secrete PDIs upon activation, which regulate platelet aggregation. However, platelets secrete only ∼10% of their PDI content extracellularly. The intracellular role of PDIs in platelet function is unknown. Here, we aim to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5floxed (fl)/fl. Pf4Cre+/ERp5fl/fl mice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a twofold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kilodalton glucose-regulated protein (GRP78), and calreticulin. ERp5-deficient platelets showed an enhanced ER stress response to ex vivo and in vivo ER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 receptor activation, and increased thrombus formation in vivo. Our results support that ERp5 acts as a negative regulator of ER stress responses in platelets and highlight the importance of a disulfide isomerase in platelet ER homeostasis. The results also indicate a previously unanticipated role of platelet ER stress in platelet secretion and thrombosis. This may have important implications for the therapeutic applications of ER stress inhibitors in thrombosis.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

Disruption of nuclear envelope integrity as a possible initiating event in tauopathies.

The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.

The Construct Structures of Psychological and Behavioral Responses to COVID-19 Pandemic in Pregnant Women.

Aim: The present study aimed to investigate the construct structure behind the psychosocial response, behavioral response, prenatal depression, and post-traumatic stress disorder (PTSD) in pregnant women during the COVID-19 pandemic in China. Method: The validated Chinese version of the Edinburgh Postnatal Depression Scale (EPDS), PTSD CheckList (PCL)-6, and two newly established scales for COVID-19-related psychological and behavioral responses were used. Structural equation modeling (SEM) analysis was applied to evaluate the structural relationships of psychological and behavioral responses during the COVID-19 pandemic. Results: Of the 1,908 mothers who completed the questionnaires, 1,099 met the criteria for perinatal depression, and 287 were positively screened for PTSD, where 264 women exceed the cut-off points for both. Pregnant women with full-time or part-time jobs tended to have the lowest scores of EPDS (10.07 ± 5.11, P < 0.001) and stress levels (23.85 ± 7.96, P = 0.004), yet they were more likely to change their behavior in accordance with the COVID-19 outbreak (13.35 ± 3.42, P = 0.025). The structural model fit the data (χ2 = 43.260, p < 0.001) and resulted in satisfactory fit indices (CFI = 0.984, TLI = 0.959, RMSEA = 0.072, and χ2/df = 10.815), all path loadings were significant (p < 0.05). The SEM indicates that the level of QoL was attributable to the occurrence of PND, leading to PTSD, and COVID-19 related behavioral and psychological responses. Conclusion: The inter-relationships between the COVID-19-related psychosocial and behavioral responses have been assessed, indicating that the pandemic increased the burden of perinatal depression. Psychoeducation, as well as other psychological interventions, may be needed to alleviate the COVID-19-based anxiety and increase their engagement in protective behaviors.

Mechano-Redox Control of Integrins in Thromboinflammation.

Significance: How mechanical forces and biochemical cues are coupled remains a miracle for many biological processes. Integrins, well-known adhesion receptors, sense changes in mechanical forces and reduction-oxidation reactions (redox) in their environment to mediate their adhesive function. The coupling of mechanical and redox function is a new area of investigation. Disturbance of normal mechanical forces and the redox balance occurs in thromboinflammatory conditions; atherosclerotic plaques create changes to the mechanical forces in the circulation. Diabetes induces redox changes in the circulation by the production of reactive oxygen species and vascular inflammation. Recent Advances: Integrins sense changes in the blood flow shear stress at the level of focal adhesions and respond to flow and traction forces by increased signaling. Talin, the integrin-actin linker, is a traction force sensor and adaptor. Oxidation and reduction of integrin disulfide bonds regulate their adhesion. A conserved disulfide bond in integrin αlpha IIb beta 3 (αIIbß3) is directly reduced by the thiol oxidoreductase endoplasmic reticulum protein 5 (ERp5) under shear stress. Critical Issues: The coordination of mechano-redox events between the extracellular and intracellular compartments is an active area of investigation. Another fundamental issue is to determine the spatiotemporal arrangement of key regulators of integrins' mechanical and redox interactions. How thromboinflammatory conditions lead to mechanoredox uncoupling is relatively unexplored. Future Directions: Integrated approaches, involving disulfide bond biochemistry, microfluidic assays, and dynamic force spectroscopy, will aid in showing that cell adhesion constitutes a crossroad of mechano- and redox biology, within the same molecule, the integrin. Antioxid. Redox Signal. 37, 1072-1093.

An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

The monoclonal antibody combination REGEN-COV protects against SARS-CoV-2 mutational escape in preclinical and human studies.

Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. Because rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting.

Influenza A Virus Hemagglutinin Is Produced in Different Disulfide-Bonded States.

Aims: Influenza A virus hemagglutinin (HA) binding to sialic acid on lung epithelial cells triggers membrane fusion and infection. Host thiol isomerases have been shown to play a role in influenza A virus infection, and we hypothesized that this role involved manipulation of disulfide bonds in HA. Results: Analysis of HA crystal structures revealed that three of the six HA disulfides occur in high-energy conformations and four of the six bonds can exist in unformed states, suggesting that the disulfide landscape of HA is generally strained and the bonds may be labile. We measured the redox state of influenza A virus HA disulfide bonds and their susceptibility to cleavage by vascular thiol isomerases. Using differential cysteine alkylation and mass spectrometry, we show that all six HA disulfide bonds exist in unformed states in ∼1 in 10 recombinant and viral surface HA molecules. Four of the six H1 and H3 HA bonds are cleaved by the vascular thiol isomerases, thioredoxin and protein disulphide isomerase, in recombinant proteins, which correlated with surface exposure of the disulfides in crystal structures. In contrast, viral surface HA disulfide bonds are impervious to five different vascular thiol isomerases. Innovation: It has been assumed that the disulfide bonds in mature HA protein are intact and inert. We show that all six HA disulfide bonds can exist in unformed states. Conclusion: These findings indicate that influenza A virus HA disulfides are naturally labile but not substrates for thiol isomerases when expressed on the viral surface.

Pathophysiological roles of cell surface and extracellular protein disulfide isomerase and their molecular mechanisms.

Protein disulfide isomerase (PDI) is the prototypic member of the thiol isomerase family that catalyses disulfide bond rearrangement. Initially identified in the endoplasmic reticulum as folding catalysts, PDI and other members in its family have also been widely reported to reside on the cell surface and in the extracellular matrix. Although how PDI is exported and retained on the cell surface remains a subject of debate, this unique pool of PDI is developing into an important mechanism underlying the redox regulation of protein sulfhydryls that are critical for the cellular activities under various disease conditions. This review aims to provide an overview of the pathophysiological roles of surface and extracellular PDI and their underlying molecular mechanisms. Understanding the involvement of extracellular PDI in these diseases will advance our knowledge in the molecular aetiology to facilitate the development of novel pharmacological strategies by specifically targeting PDI in extracellular compartments.

A New Era of Epidemiology: Digital Epidemiology for Investigating the COVID-19 Outbreak in China.

A novel pneumonia-like coronavirus disease (COVID-19) caused by a novel coronavirus named SARS-CoV-2 has swept across China and the world. Public health measures that were effective in previous infection outbreaks (eg, wearing a face mask, quarantining) were implemented in this outbreak. Available multidimensional social network data that take advantage of the recent rapid development of information and communication technologies allow for an exploration of disease spread and control via a modernized epidemiological approach. By using spatiotemporal data and real-time information, we can provide more accurate estimates of disease spread patterns related to human activities and enable more efficient responses to the outbreak. Two real cases during the COVID-19 outbreak demonstrated the application of emerging technologies and digital data in monitoring human movements related to disease spread. Although the ethical issues related to using digital epidemiology are still under debate, the cases reported in this article may enable the identification of more effective public health measures, as well as future applications of such digitally directed epidemiological approaches in controlling infectious disease outbreaks, which offer an alternative and modern outlook on addressing the long-standing challenges in population health.

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7.

The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans-differentiation approach employing natural factors may allow better control over reprogramming and improved safety. We report here a novel approach to drive trans-differentiation of human fibroblasts into functional osteoblasts using insulin-like growth factor binding protein 7 (IGFBP7). We initially determined that media conditioned by human osteoblasts can induce reprogramming of human fibroblasts to functional osteoblasts. Proteomic analysis identified IGFBP7 as being significantly elevated in media conditioned with osteoblasts compared with those with fibroblasts. Recombinant IGFBP7 induced a phenotypic switch from fibroblasts to osteoblasts. The switch was associated with senescence and dependent on autocrine IL-6 signaling. Our study supports a novel strategy for regenerating bone by using IGFBP7 to trans-differentiate fibroblasts to osteoblasts.

Autophagy protects tumors from T cell-mediated cytotoxicity via inhibition of TNFα-induced apoptosis.

Although T cell checkpoint inhibitors have transformed the treatment of cancer, the molecular determinants of tumor cell sensitivity to T cell-mediated killing need further elucidation. Here, we describe a mouse genome-scale CRISPR knockout screen that identifies tumor cell TNFα signaling as an important component of T cell-induced apoptosis, with NF-κB signaling and autophagy as major protective mechanisms. Knockout of individual autophagy genes sensitized tumor cells to killing by T cells that were activated via specific TCR or by a CD3 bispecific antibody. Conversely, inhibition of mTOR signaling, which results in increased autophagic activity, protected tumor cells from T cell killing. Autophagy functions at a relatively early step in the TNFα signaling pathway, limiting FADD-dependent caspase-8 activation. Genetic inactivation of tumor cell autophagy enhanced the efficacy of immune checkpoint blockade in mouse tumor models. Thus, targeting the protective autophagy pathway might sensitize tumors to T cell-engaging immunotherapies in the clinic.

Global redox proteome and phosphoproteome analysis reveals redox switch in Akt.

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.