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Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in MDS and AML have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in pre-clinical studies and in clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. Here, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that co-targeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. While IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complimentary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via non-canonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the PRC2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in vitro and in xenograft studies when treated with KME-2780 as compared to selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for co-targeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000-10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.

Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a PAM lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes suggested a role for osteoclast-like cells in the defense against microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activity in a receptor activator of nuclear factor-κB ligand (RANKL) and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

RATIONALE: Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. OBJECTIVE: Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. METHODS AND RESULTS: To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Single Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

Objective: It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein 1 (ABCA1). This study used proteomic analysis to identify additional cardiovascular targets of miR-144, and subsequently examined the role of a newly identified regulator of atherosclerotic burden in miR-144 KO mice receiving a high fat diet (HFD). Approach and Results: To identify affected secretory proteins, miR-144 treated endothelial cell culture medium was subjected to proteomic analysis including two-dimensional gel separation and nanospray liquid chromatography coupled to tandem mass spectrometry. We identified 5 gel spots representing 19 proteins that changed consistently across the biological replicates. One of these spots, was identified as vimentin. Atherosclerosis was induced in miR-144 KO mice by HFD and vascular lesions were quantified by Oil Red-O staining of the serial sectioned aortic root and from en-face views of the aortic tree. Unexpectedly, HFD induced extensive atherosclerosis in miR-144 KO mice in aortic root (WT 100% ± 22.71 vs miR-144 KO 256.88% ± 58.19, p < 0.05, n = 10) and arch areas (WT 100% ± 29.67 vs miR-144 KO 371.74% ± 73.48, p < 0.01, n = 10), and was accompanied by severe fatty liver disease compared with wild type littermates (WT). Vimentin levels were reduced by miR-144 and increased by antagomiR-144 in cultured cardiac endothelial cells without affecting vimentin mRNA levels. Compared with WT, ablation of the miR-144/451 cluster significantly increased plasma vimentin, while vimentin levels were decreased in control mice injected with synthetic miR-144 as double confirmed with Western blot and ELISA assays. Furthermore, increased vimentin expression was seen in the commissural regions of the aortic root (WT 100% ± 10.02 vs 164.19 ± 13.90, p < 0.01, n = 10) and collocated with other areas of aortic atherosclerosis. In contrast, neither liver ABCA1 nor circulating cholesterol levels were different between WT and miR-144/451 KO mice. Conclusion: We conclude that miR-144 is a potential regulator of the development of atherosclerosis via changes in vimentin signaling and both miR-144 and vimentin are potential therapeutic targets for atherosclerosis.

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’)2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’)2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.

SUMMARY TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is implicated in myeloid malignancies with deletion of chromosome 5q. Employing a combination of proteomic and genetic approaches, we find that TIFAB regulates ubiquitin-specific peptidase 15 (USP15) ubiquitin hydrolase activity. Expression of TIFAB in hematopoietic stem/progenitor cells (HSPCs) permits USP15 signaling to substrates, including MDM2 and KEAP1, and mitigates p53 expression. Consequently, TIFAB-deficient HSPCs exhibit compromised USP15 signaling and are sensitized to hematopoietic stress by derepression of p53. In MLL-AF9 leukemia, deletion of TIFAB increases p53 signaling and correspondingly decreases leukemic cell function and development of leukemia. Restoring USP15 expression partially rescues the function of TIFAB-deficient MLL-AF9 cells. Conversely, elevated TIFAB represses p53, increases leukemic progenitor function, and correlates with MLL gene expression programs in leukemia patients. Our studies uncover a function of TIFAB as an effector of USP15 activity and rheostat of p53 signaling in stressed and malignant HSPCs., Graphical Abstract, In Brief Niederkorn et al. identify TIFAB as a critical node in hematopoietic cells under stressed and oncogenic cell states. Their studies indicate that deregulation of the TIFAB-USP15 complex, as observed in del(5q) myelodysplasia or MLL-rearranged leukemia, modulates p53 activity and has critical functional consequences for stressed and malignant hematopoietic cells.

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8 ± 0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.

SummaryGametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.

The protein scaffold and signaling regulator p62 is important in critical cellular functions, including bone homeostasis, obesity, and cancer, because of its interactions with various signaling intermediaries. p62 is overexpressed in human cancers and is induced during cell transformation. Its genetic ablation inhibits lung tumorigenesis in vivo and cell proliferation in culture by regulating the TRAF6/NF-κB signaling cascade to control reactive oxygen species (ROS) production and apoptosis. Here we show that cdk1 phosphorylates p62 in vitro and in vivo at T269 and S272, which is necessary for the maintenance of appropriate cyclin B1 levels and the levels of cdk1 activity necessary to allow cells to properly enter and exit mitosis. The lack of cdk1-mediated phosphorylation of p62 leads to a faster exit from mitosis, which translates into enhanced cell proliferation and tumorigenesis in response to Ras-induced transformation. Therefore, p62 emerges as a node for the control of not only cell survival but also cell transit through mitosis.

The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp399 and the di-isoleucines, Ile402 and Ile403. A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs.

Individuals with clonal hematopoiesis of indeterminant potential (CHIP) are healthy, however they are at an increased risk of developing hematopoietic malignancies. The most frequent mutations in CHIP target DNMT3A and TET2, also are observed in acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndromes (MDS). These findings indicate that additional alterations are needed for the transition from a pre-leukemic stage to frank leukemia, although the identity of such molecular events remains uncharacterized. To identify cellular states that cooperate with Tet2 loss, we used in vivo RNAi screening and identified the ubiquitin ligase TRAF6 required for malignant transformation of pre-leukemic TET2-deficient hematopoietic stem/progenitor cell (HSPC). Importantly, TRAF6 expression is significantly reduced in 25-50% of AML and MPN patients as compared to healthy controls. Furthermore, TET2 mutations are more strongly correlated with lower expression of TRAF6 as compared to patients with higher TRAF6 expression in certain subsets of AML. To evaluate the consequences of TRAF6 deletion on TET2-deficienct pre-leukemic cells, we generated mice in which TRAF6 and TET2 are conditionally deleted in hematopoietic cells (VavCre;Traf6fl/fl;Tet2fl/fl[DKO]). Traf6KO mice developed a lethal phenotype with signs of MPN, including lymphopenia, neutrophilia, and increased hemoglobin levels; however, this disease was not transplantable. In striking contrast, deletion of TRAF6 in the context of TET2-deficient HSPC resulted in a rapid, penetrant, aggressive, and transplantable MPN/AML. To firmly establish that TRAF6 exhibits tumor suppressor functions, we determined whether physiological levels of TRAF6 overexpression could prevent malignant transformation. Overexpression of TRAF6 in FLT3-ITD mice inhibited malignant myeloid cell expansion in FLT3-ITD mice, and rescued the survival of the animals. To uncover the molecular basis of TRAF6's tumor suppressor function, we performed gene expression profiling and proteomic characterization of TRAF6 ubiquitination substrates in leukemic cells. RNA-sequencing of HSPC revealed that deletion of TRAF6 resulted in a significant overexpression of MYC regulated genes in pre-leukemic HSPC. In support of these findings, the proteomic screen along with extensive in vitro validation experiments identified MYC as a substrate of TRAF6. Unlike the majority of reported ubiquitin-dependent post-translational modifications of MYC, we found that ubiquitination of MYC on Lysine (K) 148 by TRAF6 does not affect its protein stability but rather antagonizes acetylation of MYC on the same lysine and thus suppresses MYC oncogenic activity. We extended these observations to investigate whether inflammatory signaling via Toll-like receptors (TLRs) can antagonize MYC function and suppress leukemic cells. Stimulation of TLRs on leukemic cells resulted in TRAF6-dependent ubiquitination of MYC at K148, which coincided with repositioning of MYC off of its target gene promoters and enhancers, and ultimately in the suppression of leukemic cell viability. Our results demonstrate that TRAF6 functions as a tumor suppressor via its ubiquitination activity that antagonizes K148 acetylation leading to a decrease of MYC transcriptional activity without affecting its protein abundance. Our findings identify TRAF6 as a novel, context-dependent tumor suppressor in myeloid neoplasms, and suggest that innate immune signaling via TLR/TRAF6 could explain why some of the clonal hematopoiesis patients develop AML and others do not. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership. Starczynowski:Kurome Therapeutics: Consultancy.

Granulocyte colony-stimulating factor is a hematopoietic cytokine that stimulates neutrophil production and hematopoietic stem cell mobilization by initiating the dimerization of homodimeric granulocyte colony-stimulating factor receptor. Different mutations of CSF3R have been linked to a unique spectrum of myeloid disorders and related malignancies. Myeloid disorders caused by the CSF3R mutations include severe congenital neutropenia, chronic neutrophilic leukemia, and atypical chronic myeloid leukemia. In this review, we provide an analysis of granulocyte colony-stimulating factor receptor, various mutations, and their roles in the severe congenital neutropenia, chronic neutrophilic leukemia, and malignant transformation, as well as the clinical implications and some perspective on approaches that could expand our knowledge with respect to the normal signaling mechanisms and those associated with mutations in the receptor.

Simple and rapid methods for analysis of monoclonal antibody structure and post-translational modifications are increasingly needed due to the explosion of therapeutic monoclonal antibodies and monoclonal antibody applications. Mass spectral analysis is a powerful method for characterizing monoclonal antibodies. Recent discovery and commercialization of the Immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS protease) has facilitated and improved the generation of antibody fragments of suitable size to allow characterization of the structure of the entire antibody molecule via analysis of just a few fragments. In this study, we coupled IdeS fragmentation and simultaneous reduction and alkylation of the resultant fragments using tributylphosphine and iodoacetamide to prepare samples in about 2 hours. Following simple introduction of a single, unseparated mixture of alkylated fragments into a mass spectrometer, detailed structural information is obtained, covering the entire antibody molecule. The large majority of the glycoforms present on the single, conserved N-linked glycosylation site of the heavy chain is elucidated, although some of the very low abundance glycoforms are not determined by this protocol. The ease, simplicity, speed, and power of this method make it attractive for analysis of the developmental stages and production batches of therapeutic monoclonal antibodies.

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.

Efforts to understand the genomic impact of human-disease-relevant genetic lesions and how they disrupt the normal sequence of cell-state transitions is hampered by a lack of defined hierarchical cellular states and corresponding networks of regulatory genes (transcription factors). Severe congenital neutropenia (SCN) patients display inherited and de novo mutations in Growth factor independent-1 (GFI1), which encodes a zinc-finger transcription factor. We identified known (e.g. N382S in zinc finger 5) and novel GFI1 sequence changes in SCN patients, then used lentiviral mediated expression to functionally evaluate them. GFI1-N382S, GFI1-K403R and GFI1-R412X mutations (in zinc finger 6) significantly elevated the expression of the Gfi1 target gene, Irf8. We generated mice with these patient-derived SCN-associated mutations in the murine Gfi1 locus. Neonatal and adult Gfi1N382S/- and Gfi1R412X/-mice are neutropenic, but Gfi1K403R/- mice have normal steady-state neutrophil levels. The resulting steady-state dysgranulopoiesis in adult mice was further pronounced in neonates. We noted that Gfi1R412X/-mice accumulate less Gfi1 protein than Gfi1+/+, while Gfi1R412X/R412Xhomozygous alleles genetically rescued both the hypomorphic protein defect and substantially restored neutrophil numbers (though not to normal). In contrast, functional challenge with neutrophil-dependent pathogens in vivo revealed a broad susceptibility for all Gfi1-mutant mice. To determine the underlying mechanism of neutropenia and immune defects, we first used novel flow cytometry analyses and Fluidigm C1 single cell RNA-Seq to establish the successive genomic states encompassing normal granulocyte specification and commitment. Independent CITE-Seq/10x sequencing analysis provided direct correlation between flow cytometry populations and genomic information, while also establishing the trajectory through genomic states traversed during terminal granulopoiesis. Next, using a novel bioinformatics algorithm (cellHarmony) we assigned Gfi1-mutant cells to their respective wild-type cell states and then determined differential gene expression. We find few genes deregulated across granulopoiesis, and that the bulk of transcriptional impact on Gfi1 target genes is specific to successive granulopoietic cell states. These insights facilitated Gfi1R412X/- Irf8+/-genetic rescue of granulocytic specification, but not post-commitment defects. We noted that a portion of Gfi1R412X/-gene deregulation unrepaired by genetic rescue was enriched for chromosome organization, proteolysis, and innate immune effectors. Electron microscopy revealed uncondensed chromatin in mature Gfi1R412X/-neutrophils while SWATH proteomics identified a loss of neutrophil granule proteins and members of the NADPH oxidase complex (potentially linking SCN with chronic granulomatous disease genes). To this end, we functionally validated impaired NADPH oxidase complex function in neutrophils from Gfi1-mutant mice. We noted Gfi1 mutant mice have consistently elevated levels of granulocyte colony stimulating factor (but not other cytokines), and so we extended our analysis of oxidative burst to GCSF-rescued human SCN patients to find profound defects; underscoring the inability of genetic or cytokine rescued specification to resolve post-commitment defects. We illustrate a work flow that can be broadly applied to molecularly dissect translationally relevant mouse models of disease, and underscores the necessity of evaluating mutations within the context of relevant cell states. Disclosures Dwivedi: Abbvie: Employment. Myers:Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Nazor:BioLegend: Employment.

In the version of this Article originally published, the authors incorrectly listed an accession code as GES90642. The correct code is GSE90642 . This has now been amended in all online versions of the Article.

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans -Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

To minimize low-quantity sample handling for protein sequencing and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a system consisting of an HPLC interfaced to an automated blotting device was used for off-line sample collection. Typically, protein digests are separated by reverse-phase HPLC and the resulting peptide fractions are pooled, concentrated, and then subjected to N-terminal sequence analysis. Obtaining unambiguous sequence from peptides derived from protein digestion at subpicomole levels requires careful sample handling to prevent loss of sample. In cases where multiple sequences are present, a secondary method such as mass spectrometry is needed to confirm the identity of the peptides. To minimize sample handling, commercial microblotting instruments have become available to deposit peptides directly onto polyvinylidene difluoride (PVDF) membrane for automated N-terminal sequence analysis. In order to adapt this technology to mass spectrometry, we investigated the use of MALDI-MS compatible membranes such as Teflon and polyethylene (PE) as the blotting media for fraction collection. Using a panel of standard peptides as well as protein digests, we demonstrate that peptides separated by capillary HPLC can be collected directly onto Teflon or PE and detected into the femtomole range. Furthermore, detailed sequence analysis could be obtained by postsource decay fragmentation spectra of individual peptides blotted onto either PE or Teflon. Due to the high sensitivity of the MALDI-MS from these membranes, it was discovered that the small amount of peptide that passed through the PVDF membrane during a collection of peptides for N-terminal sequencing was sufficient to be collected and mass analyzed from a second underlying MALDI-MS compatible membrane. Therefore, from a single HPLC separation, samples could be collected onto both PVDF for traditional N-terminal sequencing and PE or Teflon for MALDI-MS. We demonstrate the general utility of this method for sequencing peptides from a tryptic digestion at subpicomole levels and for identifying unknown proteins separated by 2-dimensional gel electrophoresis. The ability to generate both N-terminal sequence and confirmatory mass information from multiple peptides in a single separation greatly improves the reliability and the accuracy of protein characterization at subpicomole levels.

Galactosyltransferase and UDP-[3H]galactose are commonly used to identify O-linked N-acetylglucosamine (O-GlcNAc)-bearing proteins and peptides. In this report we show that immobilized Ricinus communis agglutinin I (RCA I) specifically binds in vitro galactosylated O-GlcNAc-bearing peptides, facilitating their selective isolation from complex mixtures. First, the peptide YSDSPSTST was O-GlcNAc glycosylated, galactosylated, and sialylated. Of these three glycoforms, only the one with a terminal galactose interacted with the lectin. Next, RCA I was used to isolate glycopeptides from the O-GlcNAc-bearing basic phosphoprotein (BPP) of human cytomegalovirus. BPP was overexpressed using baculovirus, [3H]galactosylated, digested with trypsin, and fractionated on RCA I. Peptides that were not galactosylated passed through the column, whereas the majority of the radiolabeled glycopeptides interacted weakly with the lectin and did not require lactose or elution. These radiolabeled peptides eluted as a broad peak with the leading edge being characterized by more hydrophobic glycopeptides and the lagging edge by less hydrophobic peptides, suggesting that the polypeptide backbone may influence the interaction with the lectin. Lactose was required to elute the remaining radiolabeled peptides, suggesting that these peptides are multiply glycosylated. The weakly interacting glycopeptides were analyzed directly by liquid chromatography/electrospray-mass spectrometry (LC/ES-MS). Glycopeptides corresponding to both of the major sites of glycosylation of BPP were identified. Thus, RCA I greatly facilitates the selective isolation of in vitro galactosylated O-GlcNAc glycopeptides from complex mixtures and substantially reduces the purification required for subsequent site-mapping by gas-phase sequencing and/or LC/ES-MS.

PKCα and PKA have crucial but opposing roles in the regulation of calcium handling within myocytes. Identification of compounds that inhibit PKCα, but not PKA, are potential therapeutic targets for the treatment of heart disease. The synthesis of indolylureas are described, and a compound displaying nanomolar inhibition towards PKCα with significant selectivity over PKA has been identified.

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.

The polo-like kinases (Plks1-5) are emerging as an important class of proteins involved in many facets of cell cycle regulation and response to DNA damage and stress. Here we show that Plk3 phosphorylates the key cell cycle protein phosphatase Cdc25A on two serine residues in its cyclinB/cdk1 docking domain and regulates its stability in response to DNA damage. We generated a Plk3 knock-out mouse and show that Cdc25A protein from Plk3-deficient cells is less susceptible to DNA damage-mediated degradation than cells with functional Plk3. We also show that absence of Plk3 correlates with loss of the G1/S cell cycle checkpoint. However, neither this compromised DNA damage checkpoint nor reduced susceptibility to proteasome-mediated degradation after DNA damage translated into a significant increase in tumor incidence in the Plk3-deficient mice.

An extracellular phosphoglycan (exPG), present in the culture medium of the promastigote form of Leishmania donovani, was purified and structurally characterized. The purification scheme included ethanol precipitation of the culture medium, anion exchange chromatography, hydrophobic chromatography on phenyl-Sepharose, and preparative polyacrylamide gel electrophoresis. Structural analysis by 1H-1H NMR, methylation linkage analysis, and glycosidase digestion revealed that the exPG consisted of the following structure: (CAP)----[PO4-6Galp beta 1-4Manp alpha 1]10-11-PO4-6Galp beta 1-4Man. The cap was found to be one of several small, neutral oligosaccharides, the most abundant of which was the trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The results indicated structural analogy to the cellular-derived lipophosphoglycan (LPG) from L. donovani. The important exceptions are a lack of the lipid anchor, the entire phosphosaccharide core, and several of the repeating disaccharide units. Although the function of exPG is presently unknown, it may play a protective role for the promastigote in the insect vector or during infection of a mammalian host.

Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. However, the study of this complex process is often hampered by the lack of a suitable cell-based model and the tools to study the biochemical events that lead to new blood vessel growth. The most widely accepted model for angiogenesis is the in vivo rat corneal model. In this model, the cornea, which is normally an avascular tissue, is stimulated to undergo angiogenesis in response to silver nitrate cauterization or to the implantation of an exogenous angiogenic agent. The physical changes associated with the new vessel growth can be readily monitored visually, but the regulated biochemical events that result in the growth and remodeling of the new vessels are much more challenging to decipher. In this report, a proteomics approach is evaluated for its utility in deciphering the biochemical events during a time course of angiogenic stimulation. At various time points post-silver nitrate cautery, corneas were harvested, solubilized, and analyzed by two-dimensional gel electrophoresis. Protein expression profiles at the various stages of angiogenesis were compared to those of control corneas. One hundred and eleven differentially-expressed proteins were identified by either matrix-assisted laser desorption/ionization-time of flight mass spectrometry or liquid chromatography-coupled electrospray ionization tandem mass spectrometry. Many of the proteins up-regulated during the angiogenesis process were identified as blood-related proteins, thus validating the development of functional blood vessels in the normally avascular tissue of the cornea. Furthermore, detection of differentially-regulated proteins in cauterized versus control tissue clearly validated the utility of a proteomics approach to study this model of angiogenesis. However, in order to get at the key regulatory proteins in the angiogenesis process, it is clear that additional scale-up and enrichment approaches will be required.

A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle.

Over the past decade, a number of nuclear and cytoplasmic proteins have been identified that are modified by single N-acetylglucosamine residues attached to the hydroxyl side chain of serines or threonines (O-GlcNAc). O-GlcNAc is a dynamic modification and therefore may act in a regulatory capacity analogous to phosphorylation. To undertake site-directed mutagenesis studies of O-GlcNAc's function, it is necessary to identify the sites of glycosylation on various proteins. The current method of site mapping, which involves galactosyltransferase labeling, generation of glycopeptides by proteolysis, purification by several rounds of HPLC, and gas-phase and manual Edman sequencing, is very tedious and requires about 10 pmol of pure, labeled glycopeptide. In this report, synthetic glycopeptides were generated and used to demonstrate that O-GlcNAc-modified peptides can be rapidly identified in complex mixtures by HPLC-coupled electrospray mass spectrometry due to the partial loss of the O-linked glycan (204 amu) at a modest orifice potential. Furthermore, the exact site of glycosylation was directly identified in the low picomole range by collision-induced dissociation (CID) of the glycopeptide after removal of the O-GlcNAc by alkaline beta-elimination. The conversion of glycosylserine to 2-aminopropenoic acid (2-ap) by beta-elimination both decreased the mass of the glycopeptide by 222 amu and resulted in a CID fragment ion representing the loss of 69 amu (2-ap) instead of 87 amu (Ser) at the position of the glycosylserine. Finally, we tested this method on an identical synthetic, alpha-linked O-GalNAc-modified peptide. Like O-GlcNAc, the O-GalNAc moiety was selectively removed at a modest orifice potential; however, the beta-elimination conditions that efficiently removed the O-GlcNAc only liberated about 20% of the O-GalNAc. We conclude that the selectivity and the sensitivity of this method will make it a powerful tool for determining the sites of O-GlcNAc modification on proteins of low abundance such as transcription factors and oncogenes.

While probing murine lymphocyte cell surfaces with bovine milk galactosyltrans-ferase, we discovered a new form of protein glycosylation in which single N-acetylglucosamine monosaccharides are O-glycosidically linked to serine or threonine moieties (O-GlcNAc) (Torres and Hart 1984). Surprisingly, O-GlcNAc is highly abundant (lymphocytes have > 1.5 × 108 molecules/cell), and is exclusively localized in nucleoplasmic and cytoplasmic compartments of the cell (Kearse and Hart 1991b). Based upon probing with galactosyltransferase (Whiteheart et al. 1989), a myriad of proteins in both the nucleus and cytoplasm are modified by O-GlcNAc. O-GlcNAc has been found in eukaryotes from yeast to man (Haltiwanger et al. 1992b; Hart et al. 1989), but has not yet been detected in prokaryotes. Identified O-GlcNAc-bearing proteins are listed in Table 1. These intracellular glycoproteins have a diverse range of functions, including transcription regulatory factors, enzymes, nuclear pore proteins, cytoskeletal proteins, and viral proteins. However, all of these glycoproteins are also phosphoproteins that form reversible multimeric complexes, depending upon their phosphorylation states, thus suggesting that O-GlcNAc may playa role in mediating/modulating regulated and reversible protein subunit interactions.