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Fibroblasts, crucial for maintaining tissue homeostasis, significantly shape the tumor microenvironment (TME). In pancreatic cancer, a highly aggressive malignancy, cancer-associated fibroblast (CAF)/extracellular matrix (ECM) units dominate the TME, influencing tumor initiation, progression, and treatment responses. Palladin, an actin-associated protein, is vital for fibroblast structural integrity and activation, playing a key role in CAF/ECM functionality. Palladin interacts with cytoskeletal proteins such as alpha-actinin (α-Act) and can therefore regulate other proteins like syndecans, modulating cytoskeletal features, cell adhesion, integrin recycling, and signaling. In this review, we propose that targeting the palladin/α-Act/syndecan interaction network could modulate CAF/ECM units, potentially shifting the TME from a tumor-promoting to a tumor-suppressive state. In silico data and reported studies to suggest that stabilizing palladin-α-Act interactions, via excess palladin, influences syndecan functions; potentially modulating integrin endocytosis via syndecan engagement with protein kinase C alpha as opposed to syndecan binding to α-Act. This mechanism can then affect the distribution of active α5β1-integrin between the plasma membrane and known intracellular vesicular compartments, thereby influencing the tumor-suppressive versus tumor-promoting functions of CAF/ECM units. Understanding these interactions offers likely future therapeutic avenues for stroma normalization in pancreatic and other cancers, aiming to inhibit tumor progression and improve future treatment outcomes., (© 2024 Wiley Periodicals LLC.)

Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-β-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-β pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer., Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-β, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer and endometrial cancer, leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated colorectal cancers, endometrial cancers, and ovarian cancers classified as TMB-high ≥10 mutations/Mb (mut/Mb) or TMB-low <10 mut/Mb. TMB was significantly highest in tumors with "POLE ExoD driver plus POLE Variant" (colorectal cancer and endometrial cancer, P < 0.001; ovarian cancer, P < 0.05). TMB increased with additional POLE variants (P < 0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the "POLE ExoD driver plus POLE Variant" tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes., Significance: Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14 V763A ) , a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14 V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse ( Med14 V769A ) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14 V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAM Q498H ) ; however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants., Competing Interests: Authors US, RA, and RS (Srinivasan) are employed by TATA Consultancy Services. SEB was a principal investigator on a research service agreement between TCS and the University of California, Berkley. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sertori, Lin, Martinez, Rana, Sharo, Kazemian, Sunderam, Andrake, Shinton, Truong, Dunbrack, Liu, Srinivasan, Brenner, Seroogy, Puck, Leonard and Wiest.)

Background: Radiation exposure of tissues is associated with inflammatory cell influx. Myeloperoxidase (MPO) is an enzyme expressed in granulocytes, such as neutrophils (PMN) and macrophages, responsible for active chlorine species (ACS) generation. The present study aimed to: 1) determine whether exposure to γ-irradiation induces MPO-dependent ACS generation in murine PMN; 2) elucidate the mechanism of radiation-induced ACS generation; and 3) evaluate the effect of the synthetic lignan LGM2605, known for ACS scavenging properties., Methods: MPO-dependent ACS generation was determined by using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF) and a highly potent MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), and confirmed in PMN derived from MPO -/- mice. Radiation-induced MPO activation was determined by EPR spectroscopy and computational analysis identified tyrosine, serine, and threonine residues near MPO's active site., Results: γ-radiation increased MPO-dependent ACS generation dose-dependently in human MPO and in wild-type murine PMN, but not in PMN from MPO -/- mice. LGM2605 decreased radiation-induced, MPO-dependent ACS. Protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSTP) inhibitors decreased the radiation-induced increase in ACS. Peroxidase cycle results demonstrate that tyrosine phosphorylation blocks MPO Compound I formation by preventing catalysis on H 2 O 2 in the active site of MPO. EPR data demonstrate that γ-radiation increased tyrosyl radical species formation in a dose-dependent manner., Conclusions: We demonstrate that γ-radiation induces MPO-dependent generation of ACS, which is dependent, at least in part, by protein tyrosine and Ser/Thr dephosphorylation and is reduced by LGM2605. This study identified for the first time a novel protein dephosphorylation-dependent mechanism of radiation-induced MPO activation., Competing Interests: Declaration of Competing Interest Melpo Christofidou-Solomidou (MCS) reports grants from the National Institutes of Health (NIH) and the National Aeronautics and Space Administration (NASA) during the conduct of the study. In addition, MCS has patents No. US 10,045,951 B2, No. US 10,030,040 B2, and No. US 9,987,321 B2 issued and patents No. PCT/US2016/049780, No. PCT/US17/35960, and No. PCT/US2008/006694 pending, and has a founders equity position in LignaMed, LLC. MCS, AVP, and MA report grants from the NIH during the conduct of the study. RAP, W-TH, ENO, and OPM have nothing to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils., Methods: MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site., Results: LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl - . EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site., Conclusions: We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Plk2 is a target of p53. Our previous studies demonstrated that with wild-type p53, Plk2 impacts mTOR signaling in the same manner as TSC1, and Plk2-deficient tumors grew larger than control. Other investigators have demonstrated that Plk2 phosphorylates mutant p53 in a positive feedback loop. We investigated Plk2's tumor suppressor functions in relationship to mTOR signaling. Archival specimens from 12 colorectal adenocarcinomas were stained for markers including Plk2, phosphorylated mTOR (serine 2448) and ribosomal S6 (Serine 235/236). We show that Plk2 is expressed in normal colon, with a punctate staining pattern in supranuclear cytoplasm. In colorectal adenocarcinoma, Plk2 demonstrates complete or partial loss of expression. Strong expression of phosphorylated mTOR is observed in the invasive front. Phosphorylated S6 expression partially correlates with phosphorylated mTOR expression but appears more diffuse in some cases. p53 and Ki67 expression is diffuse, in the subset of cases examined. In order to determine whether Plk2 is lost prior to the development of invasive cancer, 8 colon polyps from 6 patients were evaluated for Plk2 expression. All polyps are positive for Plk2. A Cancer Genome Atlas search identified Plk2 mutations to be infrequent in colorectal adenocarcinomas. Neither Plk2 methylation (in the gene body) nor copy number variations correlated with changes in mRNA expression levels. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR and phosphorylated S6 at the invasive front in some colorectal carcinomas is consistent with previous findings that an interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor suppression. Plk2 protein expression is lost at the same stage in colorectal carcinogenesis as p53. The p53 dependence of Plk2 loss and tumor suppressor function in relationship to mTOR signaling may have therapeutic implications., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P < 0.0001) in BRCA2. Of 1104 profiled CRCs from a second cohort (COSMIC), MSH2/MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P < 0.0000001). BRCA2 mutations in MSH2/MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors (P < 0.0000001). Approximately 15% of EGFR mutations found may be actionable through TKI therapy, including N700D, G719D, T725M, T790M, and E884K. NTRK gene mutations were identified in MSH2/MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Next-generation sequencing of primary and metachronous metastatic cancer lesions may impact patient care. We present a case of relapsed metastatic breast cancer with a dominant pulmonary lesion originally identified as lung adenocarcinoma. A 72-year-old, never-smoker woman with a protracted cough was found to have a large lung mass and regional lymphadenopathy on a chest CT. Lung mass biopsy showed adenocarcinoma with focal TTF-1 (thyroid transcription factor 1) positivity, favoring a lung primary. In addition to stereotactic brain radiation for cerebral metastases, she was started on carboplatin/pemetrexed. As part of the workup, the tumor was analyzed by a 50-gene targeted mutation panel, which detected 3 somatic mutations: ERBB2 (HER2) D769H activating missense mutation, TP53 Y126 inactivating truncating mutation, and SMARCB1 R374Q missense mutation. Of note, the patient had a history of stage IIA triple-negative grade 3 invasive ductal carcinoma of the left breast 1.5 years ago and received neoadjuvant chemotherapy and adjuvant radiation, and underwent a lumpectomy. Further analysis of her primary breast tumor showed a mutational profile identical to that of the lung tumor. Fluorescence in situ hybridization revealed HER2 amplification in the lung tumor, with a HER2/CEP17 ratio of 3.9. The patient was diagnosed with recurrent HER2-positive metastatic breast carcinoma with a coexisting ERBB2 (HER2) activating mutation. Chemotherapy was adjusted to include dual HER2-targeted therapy containing trastuzumab and pertuzumab, resulting in an ongoing partial response. This case demonstrates that a unique genetic mutational profile can clarify whether a tumor represents a metastatic lesion or new malignancy when conventional morphological and immunohistochemical methods are indeterminate, and can directly impact treatment decisions., (Copyright © 2015 by the National Comprehensive Cancer Network.)

Unlabelled: Many if not most proteins function in oligomeric assemblies of one or more protein sequences. The Protein Data Bank provides coordinates for biological assemblies for each entry, at least 60% of which are dimers or larger assemblies. BioAssemblyModeler (BAM) is a graphical user interface to the basic steps in homology modeling of protein homooligomers and heterooligomers from the biological assemblies provided in the PDB. BAM takes as input up to six different protein sequences and begins by assigning Pfam domains to the target sequences. The program utilizes a complete assignment of Pfam domains to sequences in the PDB, PDBfam (http://dunbrack2.fccc.edu/protcid/pdbfam), to obtain templates that contain any or all of the domains assigned to the target sequence(s). The contents of the biological assemblies of potential templates are provided, and alignments of the target sequences to the templates are produced with a profile-profile alignment algorithm. BAM provides for visual examination and mouse-editing of the alignments supported by target and template secondary structure information and a 3D viewer of the template biological assembly. Side-chain coordinates for a model of the biological assembly are built with the program SCWRL4. A built-in protocol navigation system guides the user through all stages of homology modeling from input sequences to a three-dimensional model of the target complex., Availability: http://dunbrack.fccc.edu/BAM.

Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.

The structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ∼90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the intersubunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins., Methodology/results: Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate., Conclusion: Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure-function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.

Sophisticated models for the regulation of mitotic entry are lacking for human cells. Inactivating human cyclin A/Cdk2 complexes through diverse approaches delays mitotic entry and promotes inhibitory phosphorylation of Cdk1 on tyrosine 15, a modification performed by Wee1. We show here that cyclin A/Cdk2 complexes physically associate with Wee1 in U2OS cells. Mutation of four conserved RXL cyclin A/Cdk binding motifs (RXL1 to RXL4) in Wee1 diminished stable binding. RXL1 resides within a large regulatory region of Wee1 that is predicted to be intrinsically disordered (residues 1 to 292). Near RXL1 is T239, a site of inhibitory Cdk phosphorylation in Xenopus Wee1 proteins. We found that T239 is phosphorylated in human Wee1 and that this phosphorylation was reduced in an RXL1 mutant. RXL1 and T239 mutants each mediated greater Cdk phosphorylation and G(2)/M inhibition than the wild type, suggesting that cyclin A/Cdk complexes inhibit human Wee1 through these sites. The RXL1 mutant uniquely also displayed increased nuclear localization. RXL1 is embedded within sequences homologous to Crm1-dependent nuclear export signals (NESs). Coimmunoprecipitation showed that Crm1 associated with Wee1. Moreover, treatment with the Crm1 inhibitor leptomycin B or independent mutation of the potential NES (NESm) abolished Wee1 nuclear export. Export was also reduced by Cdk inhibition or cyclin A RNA interference, suggesting that cyclin A/Cdk complexes contribute to Wee1 export. Somewhat surprisingly, NESm did not display increased G(2)/M inhibition. Thus, nuclear export of Wee1 is not essential for mitotic entry though an important functional role remains likely. These studies identify a novel bifunctional regulatory element in Wee1 that mediates cyclin A/Cdk2 association and nuclear export.

Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571-3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.