A signaling inspired synthetic toolkit for efficient production of tyrosine phosphorylated proteins.

Tyrosine phosphorylation is an important post-translational modification that regulates many biochemical signaling networks in multicellular organisms. To date, 46,000 tyrosines have been observed in human proteins, but relatively little is known about the function and regulation of most of these sites. A major challenge has been producing recombinant phospho-proteins in order to test the effects of phosphorylation.

Quantitative proteomic mass spectrometry of protein kinases to determine dynamic heterogeneity of the human kinome.

The kinome is a dynamic system of kinases regulating signaling networks in cells and dysfunction of protein kinases contributes to many diseases. Regulation of the protein expression of kinases alters cellular responses to environmental changes and perturbations. We configured a library of 672 proteotypic peptides to quantify >300 kinases in a single LC-MS experiment using ten micrograms protein from human tissues including biopsies.

HTLV-1 infected T cells cause bone loss via small extracellular vesicles.

Adult T cell leukaemia (ATL), caused by infection with human T- lymphotropic virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T leads to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone.

Missense Mutations in Myc Box I Influence Nucleocytoplasmic Transport to Promote Leukemogenesis.

Purpose: Somatic missense mutations in the phosphodegron domain of the MYC gene (MYC Box I or MBI) are detected in the dominant clones of a subset of patients with acute myeloid leukemia (AML), but the mechanisms by which they contribute to AML are unknown.

Experimental Design: To investigate the effects of MBI MYC mutations on hematopoietic cells, we employed a multi-omic approach to systematically compare the cellular and molecular consequences of expressing oncogenic doses of wild type, threonine-58 and proline-59 mutant MYC proteins in hematopoietic cells, and we developed a knockin mouse harboring the germline MBI mutation p.T58N in the Myc gene.

HTLV-1 infected T cells cause bone loss via small extracellular vesicles.

Adult T cell leukemia (ATL), caused by infection with human T cell leukemia virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T lead to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone.

Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined.

Missense mutations in Myc Box I influence MYC cellular localization, mRNA partitioning and turnover to promote leukemogenesis.

Somatic missense mutations in the phosphodegron domain of the gene ( M YC Box I) are detected in the dominant clones of a subset of acute myeloid leukemia (AML) patients, but the mechanisms by which they contribute to AML are unknown. To unveil unique proprieties of MBI MYC mutant proteins, we systematically compared the cellular and molecular consequences of expressing similar oncogenic levels of wild type and MBI mutant MYC. We found that MBI MYC mutants can accelerate leukemia by driving unique transcriptional signatures in highly selected, myeloid progenitor subpopulations.

Identification of biomarker candidates for filarial parasite infections by analysis of extracellular vesicles.

Background: Improved diagnostic tools are needed for detecting active filarial infections in humans. Tests are available that detect adult circulating filarial antigen, but there are no sensitive and specific biomarker tests for brugian filariasis or loiasis. Here we explored whether extracellular vesicles released by filarial parasites contain diagnostic biomarker candidates.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC).

The proteome of extracellular vesicles of the lung fluke Paragonimus kellicotti produced in vitro and in the lung cyst.

Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by Paragonimus kellicotti adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils.

Sleep deprivation exacerbates microglial reactivity and Aβ deposition in a -dependent manner in mice.

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer's disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-β (Aβ) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression.

Highly sensitive detection of dynamic changes in enkephalins following acute stress.

Enkephalins are opioid peptides that modulate analgesia, reward, and stress. detection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani, 2018, eLife).

Longitudinal study of cross-reactive antigenemia in individuals with high microfilarial density reveals promising biomarkers for distinguishing lymphatic filariasis from loiasis.

Background And Methods: Circulating antigens are often detected in individuals with heavy infections by diagnostic tests for lymphatic filariasis (LF) caused by . This is a major challenge to LF mapping and elimination efforts in loiasis co-endemic areas. However, it also provides an opportunity to identify antigen biomarkers for loiasis.

Comparative proteomics of adult Paragonimus kellicotti excretion/secretion products released in vitro or present in the lung cyst nodule.

Paragonimus kellicotti is a zoonotic lung fluke infection, the agent of North American paragonimiasis, and an excellent model for other Paragonimus infections. The excretory/secretory proteins (ESP) released by parasites and presented at the parasite-host interface are frequently proposed to be useful targets for drugs and/or vaccines In vitro culture conditions may alter ESP compared to those produced in vivo. In order to investigate ESPs produced in vivo we took advantage of the fact that adult P.

Proteomic and phosphoproteomic landscapes of acute myeloid leukemia.

We have developed a deep-scale proteome and phosphoproteome database from 44 representative acute myeloid leukemia (AML) patients from the LAML TCGA dataset and 6 healthy bone marrow-derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of posttranscriptionally regulated proteins both globally (ie, in all AML samples) and also in patients with recurrent AML driver mutations.

TNK2/ACK1-mediated phosphorylation of ATP5F1A (ATP synthase F1 subunit alpha) selectively augments survival of prostate cancer while engendering mitochondrial vulnerability.

The challenge of rapid macromolecular synthesis enforces the energy-hungry cancer cell mitochondria to switch their metabolic phenotypes, accomplished by activation of oncogenic tyrosine kinases. Precisely how kinase activity is directly exploited by cancer cell mitochondria to meet high-energy demand, remains to be deciphered. Here we show that a non-receptor tyrosine kinase, TNK2/ACK1 (tyrosine kinase non receptor 2), phosphorylated ATP5F1A (ATP synthase F1 subunit alpha) at Tyr243 and Tyr246 (Tyr200 and 203 in the mature protein, respectively) that not only increased the stability of complex V, but also increased mitochondrial energy output in cancer cells.

Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer.

Purpose: Androgen receptor (AR) antagonism is exacerbated by HOXB13 in castration-resistant prostate cancers (CRPC). However, it is unclear when and how HOXB13 primes CRPCs for AR antagonism. By mass-spectrometry analysis of CRPC extract, we uncovered a novel lysine 13 (K13) acetylation in HOXB13 mediated by CBP/p300.

Enterically derived high-density lipoprotein restrains liver injury through the portal vein.

The biogenesis of high-density lipoprotein (HDL) requires apoA1 and the cholesterol transporter ABCA1. Although the liver generates most of the HDL in the blood, HDL synthesis also occurs in the small intestine. Here, we show that intestine-derived HDL traverses the portal vein in the HDL subspecies form, in complex with lipopolysaccharide (LPS)-binding protein (LBP).

Chaperonin Containing TCP-1 Protein Level in Breast Cancer Cells Predicts Therapeutic Application of a Cytotoxic Peptide.

Purpose: Metastatic disease is a leading cause of death for patients with breast cancer, driving the need for new therapies. CT20p is a peptide previously discovered by our group that displays cancer-specific cytotoxicity. To design the optimal therapeutic use of the peptide, we identified the intracellular target of CT20p in breast cancer cells, correlating expression patterns of the target with susceptibility to CT20p.

Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.

Early cancers are avascular and hence, profoundly acidic. Pre-malignant cells must adapt to acidosis to thrive in this hostile microenvironment. Here, we investigate MCF-7 cells that are adapted to grow in acidic conditions using SILAC proteomics and we reveal a significant upregulation of lysosomal proteins.

Quantification of peptides from immunoglobulin constant and variable regions by LC-MRM MS for assessment of multiple myeloma patients.

Purpose: Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM).

Experimental Design: Using LC-MS/MS data, Ig constant region peptides, and transitions were selected for LC-MRM MS. Quantitative assays were used to assess Igs in serum from 83 patients.

Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash.

Background: Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake.

Methods: Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit.