Fibrosis induced by resident macrophages has divergent roles in pancreas inflammatory injury and PDAC.

Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages. Whether TRMs and monocyte-derived macrophages have district roles under differing pathologies is not understood. Here, we showed that a substantial portion of the macrophages that accumulated during pancreatitis and pancreatic cancer in mice had expanded from TRMs. Pancreas TRMs had an extracellular matrix remodeling phenotype that was important for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and death, due to impaired acinar cell survival and recovery. During pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.

Structural insights into Ubr1-mediated N-degron polyubiquitination.

The N-degron pathway targets proteins that bear a destabilizing residue at the N terminus for proteasome-dependent degradation1. In yeast, Ubr1-a single-subunit E3 ligase-is responsible for the Arg/N-degron pathway2. How Ubr1 mediates the initiation of ubiquitination and the elongation of the ubiquitin chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here we developed chemical strategies to mimic the reaction intermediates of the first and second ubiquitin transfer steps, and determined the cryo-electron microscopy structures of Ubr1 in complex with Ubc2, ubiquitin and two N-degron peptides, representing the initiation and elongation steps of ubiquitination. Key structural elements, including a Ubc2-binding region and an acceptor ubiquitin-binding loop on Ubr1, were identified and characterized. These structures provide mechanistic insights into the initiation and elongation of ubiquitination catalysed by Ubr1.

Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression.

Tumor-associated macrophages (TAMs) are essential components of the cancer microenvironment and play critical roles in the regulation of tumor progression. Optimal therapeutic intervention requires in-depth understanding of the sources that sustain macrophages in malignant tissues. In this study, we investigated the ontogeny of TAMs in murine pancreatic ductal adenocarcinoma (PDAC) models. We identified both inflammatory monocytes and tissue-resident macrophages as sources of TAMs. Unexpectedly, significant portions of pancreas-resident macrophages originated from embryonic development and expanded through in situ proliferation during tumor progression. Whereas monocyte-derived TAMs played more potent roles in antigen presentation, embryonically derived TAMs exhibited a pro-fibrotic transcriptional profile, indicative of their role in producing and remodeling molecules in the extracellular matrix. Collectively, these findings uncover the heterogeneity of TAM origin and functions and could provide therapeutic insight for PDAC treatment.

Thioester-Assisted Sortase-A-Mediated Ligation.

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a C-terminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N- or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

Potassium channel selectivity filter dynamics revealed by single-molecule FRET.

Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High-resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+ ions, but not Na+ ions, are perfectly coordinated. Using single-molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentration. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels nonselective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild-type KirBac1.1 channels are K+ selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, whereas K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

Chemical Synthesis of Activity-Based E2-Ubiquitin Probes for the Structural Analysis of E3 Ligase-Catalyzed Transthiolation.

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.

An E1-Catalyzed Chemoenzymatic Strategy to Isopeptide-N-Ethylated Deubiquitylase-Resistant Ubiquitin Probes.

Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.

Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones.

Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

Efficient Semi-Synthesis of Atypical Ubiquitin Chains and Ubiquitin-Based Probes Forged by Thioether Isopeptide Bonds.

The development of powerful and general methods to acquire ubiquitin (Ub) chains has prompted the deciphering of Ub-mediated processes. Herein, the cysteine-aminoethylation assisted chemical ubiquitination (CAACU) strategy is extended and improved to enable the efficient semi-synthesis of atypical Ub chain analogues and Ub-based probes. Combining the Cys aminoethylation and the auxiliary-mediated protein ligation, several linkage- and length-defined atypical Ub chains including di-Ubs, K27C-linked tri-Ub, K11/K48C-branched tri-Ub, and even the SUMOlated Ub are successfully prepared from recombinantly expressed starting materials at about a 9-20 mg L-1 expression level. In addition, the utility of this strategy is demonstrated with the synthesis of a novel non-hydrolyzable di-Ub PA probe, which may provide a new useful tool for the mechanistic studies of deubiquitinase (DUB) recognition.

One-pot multi-segment condensation strategies for chemical protein synthesis.

With the growing requirement for otherwise-difficult-to-obtain proteins, it is necessary to develop more efficient chemical protein synthesis methods for rapid access to designed protein samples. In particular, a one-pot multi-segment condensation method, with only one purification step to obtain the final product, is expected to demonstrate unique benefits in chemical protein synthesis, such as the requirement of fewer handling procedures and the higher efficiency in obtaining aimed protein samples. The utilization of the one-pot multi-segment condensation strategy is demonstrated via the synthesis of a series of post-translational modification (PTM) or disease-associated peptides or proteins for basic and advanced scientific research. This review summarizes the recent one-pot multi-segment condensation methods utilized in chemical protein synthesis, in which two aspects of drive-strategies will be mainly included: a kinetically controlled strategy and a protecting group-removal strategy, respectively. On one hand, the activities of peptides in N-terminal thiol amino acids or C-terminal acyl donors can be largely different based on the differences in properties, such as steric hindrance, migration rates, electrophilicity, and introduction of active elements such as selenium, etc. Using the different activities, regio-selective peptide ligation can be performed in a kinetically controlled manner. On the other hand, the protecting group-removal strategy involves various moieties, which can block the activity of functional groups arising from N-terminal thiol amino acids or C-terminal acyl donors, and they can be removed by using additives, and pH- or photo-stimulation conditions with further achievement of chemical protein synthesis by the one-pot strategy.

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase.

During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase (Hin-Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance-improving strategies, such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.

A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli.

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6-10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme.

Salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers selectively suppress human CD20+ melanoma stem cells.

Melanoma is the deadliest type of skin cancer. CD20+ melanoma stem cells (CSCs) are pivotal for metastasis and initiation of melanoma. Therefore, selective elimination of CD20+ melanoma CSCs represents an effective treatment to eradicate melanoma. Salinomycin has emerged as an effective drug toward various CSCs. Due to its poor solubility, its therapeutic efficacy against melanoma CSCs has never been evaluated. In order to target CD20+ melanoma CSCs, we designed salinomycin-loaded lipid-polymer nanoparticles with anti-CD20 aptamers (CD20-SA-NPs). Using a single-step nanoprecipitation method, salinomycin-loaded lipid-polymer nanoparticles (SA-NPs) were prepared, then CD20-SA-NPs were obtained through conjugation of thiolated anti-CD20 aptamers to SA-NPs via a maleimide-thiol reaction. CD20-SA-NPs displayed a small size of 96.3 nm, encapsulation efficiency higher than 60% and sustained drug release ability. The uptake of CD20-SA-NPs by CD20+ melanoma CSCs was significantly higher than that of SA-NPs and salinomycin, leading to greatly enhanced cytotoxic effects in vitro, thus the IC50 values of CD20-SA-NPs were reduced to 5.7 and 2.6 µg/mL in A375 CD+20 cells and WM266-4 CD+ cells, respectively. CD20-SA-NPs showed a selective cytotoxicity toward CD20+ melanoma CSCs, as evidenced by the best therapeutic efficacy in suppressing the formation of tumor spheres and the proportion of CD20+ cells in melanoma cell lines. In mice bearing melanoma xenografts, administration of CD20-SA-NPs (salinomycin 5 mg·kg-1·d-1, iv, for 60 d) showed a superior efficacy in inhibition of melanoma growth compared with SA-NPs and salinomycin. In conclusion, CD20 is a superior target for delivering drugs to melanoma CSCs. CD20-SA-NPs display effective delivery of salinomycin to CD20+ melanoma CSCs and represent a promising treatment for melanoma.

Hnf4 activates mimetic-cell enhancers to recapitulate gut and liver development within the thymus.

Mimetic cells are medullary thymic epithelial cells (mTECs) that mimic extra-thymic cell types to tolerize T cells to self-antigens. Here, we dissected the biology of entero-hepato mTECs, mimetic cells expressing gut- and liver-associated transcripts. Entero-hepato mTECs conserved their thymic identity yet accessed wide swaths of enterocyte chromatin and transcriptional programs via the transcription factors Hnf4α and Hnf4γ. Deletion of Hnf4α and Hnf4γ in TECs ablated entero-hepato mTECs and downregulated numerous gut- and liver-associated transcripts, with a primary contribution from Hnf4γ. Loss of Hnf4 impaired enhancer activation and CTCF redistribution in mTECs but did not impact Polycomb-mediated repression or promoter-proximal histone marks. By single-cell RNA sequencing, Hnf4 loss produced three distinct effects on mimetic cell state, fate, and accumulation. Serendipitously, a requirement for Hnf4 in microfold mTECs was discovered, which exposed a requirement for Hnf4γ in gut microfold cells and the IgA response. Study of Hnf4 in entero-hepato mTECs thus revealed mechanisms of gene control in the thymus and periphery alike.

A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system.

Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.

Context-dependent activation of STING-interferon signaling by CD11b agonists enhances anti-tumor immunity.

Chronic activation of inflammatory pathways and suppressed interferon are hallmarks of immunosuppressive tumors. Previous studies have shown that CD11b integrin agonists could enhance anti-tumor immunity through myeloid reprograming, but the underlying mechanisms remain unclear. Herein we find that CD11b agonists alter tumor-associated macrophage (TAM) phenotypes by repressing NF-κB signaling and activating interferon gene expression simultaneously. Repression of NF-κB signaling involves degradation of p65 protein and is context independent. In contrast, CD11b agonism induces STING/STAT1 pathway-mediated interferon gene expression through FAK-mediated mitochondrial dysfunction, with the magnitude of induction dependent on the tumor microenvironment and amplified by cytotoxic therapies. Using tissues from phase I clinical studies, we demonstrate that GB1275 treatment activates STING and STAT1 signaling in TAMs in human tumors. These findings suggest potential mechanism-based therapeutic strategies for CD11b agonists and identify patient populations more likely to benefit.

Stromal and therapy-induced macrophage proliferation promotes PDAC progression and susceptibility to innate immunotherapy.

Tumor-associated macrophages (TAMs) are abundant in pancreatic ductal adenocarcinomas (PDACs). While TAMs are known to proliferate in cancer tissues, the impact of this on macrophage phenotype and disease progression is poorly understood. We showed that in PDAC, proliferation of TAMs could be driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression also drove response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Background: Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. Methods: We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Results: Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Conclusions: Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

Stromal Reprogramming by FAK Inhibition Overcomes Radiation Resistance to Allow for Immune Priming and Response to Checkpoint Blockade.

The effects of radiotherapy (RT) on tumor immunity in pancreatic ductal adenocarcinoma (PDAC) are not well understood. To better understand if RT can prime antigen-specific T-cell responses, we analyzed human PDAC tissues and mouse models. In both settings, there was little evidence of RT-induced T-cell priming. Using in vitro systems, we found that tumor-stromal components, including fibroblasts and collagen, cooperate to blunt RT efficacy and impair RT-induced interferon signaling. Focal adhesion kinase (FAK) inhibition rescued RT efficacy in vitro and in vivo, leading to tumor regression, T-cell priming, and enhanced long-term survival in PDAC mouse models. Based on these data, we initiated a clinical trial of defactinib in combination with stereotactic body RT in patients with PDAC (NCT04331041). Analysis of PDAC tissues from these patients showed stromal reprogramming mirroring our findings in genetically engineered mouse models. Finally, the addition of checkpoint immunotherapy to RT and FAK inhibition in animal models led to complete tumor regression and long-term survival. SIGNIFICANCE: Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy. This article is highlighted in the In This Issue feature, p. 2711.