Development of an Orally Bioavailable LCK PROTAC Degrader as a Potential Therapeutic Approach to T-Cell Acute Lymphoblastic Leukemia.

Despite significant advances over recent years, the treatment of T cell acute lymphoblastic leukemia (T-ALL) remains challenging. We have recently shown that a subset of T-ALL cases exhibited constitutive activation of the lymphocyte-specific protein tyrosine kinase (LCK) and were consequently responsive to treatments with LCK inhibitors and degraders such as dasatinib and dasatinib-based PROTACs. Here we report the design, synthesis and in vitro/vivo evaluation of SJ45566, a potent and orally bioavailable LCK PROTAC.

A Study on the Maximum Reliability of Multi-UAV Cooperation Relay Systems.

This paper studies the maximum reliability of multi-hop relay UAVs, in which UAVs provide wireless services for remote users as a coded cooperative relay without an end-to-end direct communication link. In this paper, the analytical expressions of the total power loss and total bit error rate are derived as reliability measures. First, based on the environmental statistical parameters, a LOS probability model is proposed. Then, the problem of minimizing the bit error rate of static and mobile UAVs is studied. The goal is to minimize the total bit error rate by jointly optimizing the height, elevation, power and path loss and introducing the maximum allowable path loss constraints, transmission power allocation constraints, and UAV height and elevation constraints. At the same time, the total path loss is minimized to achieve maximum ground communication coverage. However, the formulated joint optimization problem is nonconvex and generally difficult to solve. Therefore, we decomposed the problem into two subproblems and proposed an effective joint optimization iteration algorithm. Finally, the simulation results are given, and the analysis shows that the optimal height of different reliability measures is slightly different; thus, using the mobility of UAVs can improve the reliability of communication performance.

Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons.

Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we report a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and brain LC3-interactome reveals roles of neuronal autophagy in targeting proteins of multiple cellular organelles/pathways, including endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) for degradation. By combining phosphoproteomics and functional analysis in human and mouse neurons, we uncovered a function of neuronal autophagy in controlling cAMP-PKA and c-FOS-mediated neuronal activity through selective degradation of the protein kinase A - cAMP-binding regulatory (R)-subunit I (PKA-RI) complex. Lack of AKAP11 causes accumulation of the PKA-RI complex in the soma and neurites, demonstrating a constant clearance of PKA-RI complex through AKAP11-mediated degradation in neurons. Our study thus reveals the landscape of autophagy degradation in human neurons and identifies a physiological function of autophagy in controlling homeostasis of PKA-RI complex and specific PKA activity in neurons.

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

In-Depth Blood Proteome Profiling by Extensive Fractionation and Multiplexed Quantitative Mass Spectrometry.

Blood in the circulatory system carries information of physiological and pathological status of the human body, so blood proteins are often used as biomarkers for diagnosis, prognosis, and therapy. Human blood proteome can be explored by the latest technologies in mass spectrometry (MS), creating an opportunity of discovering new disease biomarkers. The extreme dynamic range of protein concentrations in blood, however, poses a challenge to detect proteins of low abundance, namely, tissue leakage proteins. Here, we describe a strategy to directly analyze undepleted blood samples by extensive liquid chromatography (LC) fractionation and 18-plex tandem-mass-tag (TMT) mass spectrometry. The proteins in blood specimens (e.g., plasma or serum) are isolated by acetone precipitation and digested into peptides. The resulting peptides are TMT-labeled, separated by basic pH reverse-phase (RP) LC into at least 40 fractions, and analyzed by acidic pH RPLC and high-resolution MS/MS, leading to the quantification of ~3000 unique proteins. Further increase of basic pH RPLC fractions and adjustment of the fraction concatenation strategy can enhance the proteomic coverage (up to ~5000 proteins). Finally, the combination of multiple batches of TMT experiments allows the profiling of hundreds of blood samples. This TMT-MS-based method provides a powerful platform for deep proteome profiling of human blood samples.

Multi-omics analysis reveals expression complexity and functional diversity of mouse kinome.

Protein kinases are a crucial component of signaling pathways involved in a wide range of cellular responses, including growth, proliferation, differentiation, and migration. Systematic investigation of protein kinases is critical to better understand phosphorylation-mediated signaling pathways and may provide insights into the development of potential therapeutic drug targets. Here we perform a systems-level analysis of the mouse kinome by analyzing multi-omics data. We used bulk and single-cell transcriptomic data from the C57BL/6J mouse strain to define tissue- and cell-type-specific expression of protein kinases, followed by investigating variations in sequence and expression between C57BL/6J and DBA/2J strains. We then profiled a deep brain phosphoproteome from C57BL/6J and DBA/2J strains as well as their reciprocal hybrids to infer the activity of the mouse kinome. Finally, we performed phenome-wide association analysis using the BXD recombinant inbred (RI) mice (a cross between C57BL/6J and DBA/2J strains) to identify any associations between variants in protein kinases and phenotypes. Collectively, our study provides a comprehensive analysis of the mouse kinome by investigating genetic sequence variation, tissue-specific expression patterns, and associations with downstream phenotypes.

Genetic architecture of protein expression and its regulation in the mouse brain.

BACKGROUND: Natural variation in protein expression is common in all organisms and contributes to phenotypic differences among individuals. While variation in gene expression at the transcript level has been extensively investigated, the genetic mechanisms underlying variation in protein expression have lagged considerably behind. Here we investigate genetic architecture of protein expression by profiling a deep mouse brain proteome of two inbred strains, C57BL/6 J (B6) and DBA/2 J (D2), and their reciprocal F1 hybrids using two-dimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) technology. RESULTS: By comparing protein expression levels in the four mouse strains, we observed 329 statistically significant differentially expressed proteins between the two parental strains and characterized the genetic basis of protein expression. We further applied a proteogenomic approach to detect variant peptides and define protein allele-specific expression (pASE), identifying 33 variant peptides with cis-effects and 17 variant peptides showing trans-effects. Comparison of regulation at transcript and protein levels show a significant divergence. CONCLUSIONS: The results provide a comprehensive analysis of genetic architecture of protein expression and the contribution of cis- and trans-acting regulatory differences to protein expression.

Deep Multilayer Brain Proteomics Identifies Molecular Networks in Alzheimer's Disease Progression.

Alzheimer's disease (AD) displays a long asymptomatic stage before dementia. We characterize AD stage-associated molecular networks by profiling 14,513 proteins and 34,173 phosphosites in the human brain with mass spectrometry, highlighting 173 protein changes in 17 pathways. The altered proteins are validated in two independent cohorts, showing partial RNA dependency. Comparisons of brain tissue and cerebrospinal fluid proteomes reveal biomarker candidates. Combining with 5xFAD mouse analysis, we determine 15 Aß-correlated proteins (e.g., MDK, NTN1, SMOC1, SLIT2, and HTRA1). 5xFAD shows a proteomic signature similar to symptomatic AD but exhibits activation of autophagy and interferon response and lacks human-specific deleterious events, such as downregulation of neurotrophic factors and synaptic proteins. Multi-omics integration prioritizes AD-related molecules and pathways, including amyloid cascade, inflammation, complement, WNT signaling, TGF-ß and BMP signaling, lipid metabolism, iron homeostasis, and membrane transport. Some Aß-correlated proteins are colocalized with amyloid plaques. Thus, the multilayer omics approach identifies protein networks during AD progression.

Deep multiomics profiling of brain tumors identifies signaling networks downstream of cancer driver genes.

High throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors. Pathway activity computation and mouse survival indicate the NTRK1 mutation induces a higher activation of AKT downstream targets including MYC and JUN, drives a positive feedback loop to up-regulate multiple other RTKs, and confers higher oncogenic potency than the PDGFRA mutation. A mini-gRNA library CRISPR-Cas9 validation screening shows 56% of tested master regulators are important for the viability of NTRK-driven HGG cells, including TFs (Myc and Jun) and metabolic kinases (AMPKa1 and AMPKa2), confirming the validity of the multiomics integrative approaches, and providing novel tumor vulnerabilities.

Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH.

The expression of the phospholipase A2 activity (aiPLA2) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA2 assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA2 activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA2 activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H2O2 or the presence of oxidized phospholipid as substrate also significantly increased aiPLA2 activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA2 activity at pH 7 without effect on the activity of the enzyme at pH 4.

Target-Decoy-Based False Discovery Rate Estimation for Large-Scale Metabolite Identification.

Metabolite identification is a crucial step in mass spectrometry (MS)-based metabolomics. However, it is still challenging to assess the confidence of assigned metabolites. We report a novel method for estimating the false discovery rate (FDR) of metabolite assignment with a target-decoy strategy, in which the decoys are generated through violating the octet rule of chemistry by adding small odd numbers of hydrogen atoms. The target-decoy strategy was integrated into JUMPm, an automated metabolite identification pipeline for large-scale MS analysis and was also evaluated with two other metabolomics tools, mzMatch and MZmine 2. The reliability of FDR calculation was examined by false data sets, which were simulated by altering MS1 or MS2 spectra. Finally, we used the JUMPm pipeline coupled to the target-decoy strategy to process unlabeled and stable-isotope-labeled metabolomic data sets. The results demonstrate that the target-decoy strategy is a simple and effective method for evaluating the confidence of high-throughput metabolite identification.

Wearable Device-Based Gait Recognition Using Angle Embedded Gait Dynamic Images and a Convolutional Neural Network.

The widespread installation of inertial sensors in smartphones and other wearable devices provides a valuable opportunity to identify people by analyzing their gait patterns, for either cooperative or non-cooperative circumstances. However, it is still a challenging task to reliably extract discriminative features for gait recognition with noisy and complex data sequences collected from casually worn wearable devices like smartphones. To cope with this problem, we propose a novel image-based gait recognition approach using the Convolutional Neural Network (CNN) without the need to manually extract discriminative features. The CNN's input image, which is encoded straightforwardly from the inertial sensor data sequences, is called Angle Embedded Gait Dynamic Image (AE-GDI). AE-GDI is a new two-dimensional representation of gait dynamics, which is invariant to rotation and translation. The performance of the proposed approach in gait authentication and gait labeling is evaluated using two datasets: (1) the McGill University dataset, which is collected under realistic conditions; and (2) the Osaka University dataset with the largest number of subjects. Experimental results show that the proposed approach achieves competitive recognition accuracy over existing approaches and provides an effective parametric solution for identification among a large number of subjects by gait patterns.

JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells.

Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by re-searching only unmatched high-quality spectra and reusing amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then, the JUMPg program is applied to process a label-free mass spectrometry data set of Alzheimer's disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and "non-coding gene" translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled data set of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multiomics data integration.

A novel lysophosphatidylcholine acyl transferase activity is expressed by peroxiredoxin 6.

The phospholipase A2(PLA2) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation wereKm18 µM andVmax30 nmol/min/mg protein; theVmaxwas increased 25-fold by phosphorylation of the protein whileKmwas unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA2activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.

Peroxiredoxin 6 homodimerization and heterodimerization with glutathione S-transferase pi are required for its peroxidase but not phospholipase A2 activity.

Peroxiredoxin 6 (Prdx6) is a unique 1-Cys member of the peroxiredoxin family with both GSH peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by its heterodimerization with GSH S-transferase π (πGST) is required for its peroxidatic catalytic cycle. Recombinant human Prdx6 crystallizes as a homodimer and sedimentation equilibrium analysis confirmed that this protein exists as a high affinity dimer in solution. Based on measurement of molecular mass, dimeric Prdx6 that was oxidized to the sulfenic acid formed a sulfenylamide during storage. After examination of the dimer interface in the crystal structure, we postulated that the hydrophobic amino acids L145 and L148 play an important role in homodimerization of Prdx6 as well as in its heterodimerization with πGST. Oxidation of Prdx6 also was required for its heterodimerization. Sedimentation equilibrium analysis and the Duolink proximity ligation assay following mutation of the L145 and L148 residues of Prdx6 to Glu indicated greatly decreased dimerization propensity reflecting the loss of hydrophobic interactions between the protein monomers. Peroxidase activity was markedly reduced by mutation at either of the Leu sites and was essentially abolished by the double mutation, while PLA2 activity was unaffected. Decreased peroxidase activity following mutation of the interfacial leucines presumably is mediated via impaired heterodimerization of Prdx6 with πGST that is required for reduction and re-activation of the oxidized enzyme.

Mutation of Serine 32 to Threonine in Peroxiredoxin 6 Preserves Its Structure and Enzymatic Function but Abolishes Its Trafficking to Lamellar Bodies.

Peroxiredoxin 6 (Prdx6), a bifunctional protein with phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid turnover. Prdx6 has been localized to both cytosol and lamellar bodies (LB) in lung epithelium, and its organellar targeting sequence has been identified. We propose that Prdx6 LB targeting facilitates its role in the metabolism of lung surfactant phosphatidylcholine (PC). Ser-32 has been identified as the active site in Prdx6 for aiPLA2 activity, and this activity was abolished by the mutation of serine 32 to alanine (S32A). However, aiPLA2 activity was unaffected by mutation of serine 32 in Prdx6 to threonine (S32T). Prdx6 protein expression and aiPLA2 activity were normal in the whole lung of a "knock-in" mouse model carrying an S32T mutation in the Prdx6 gene but were absent from isolated LB. Analyses by proximity ligation assay in lung sections demonstrated the inability of S32T Prdx6 to bind to the chaperone protein, 14-3-3ϵ, that is required for LB targeting. The content of total phospholipid, PC, and disaturated PC in lung tissue homogenate, bronchoalveolar lavage fluid, and lung LB was increased significantly in Prdx6-S32T mutant lungs, whereas degradation of internalized [(3)H]dipalmitoyl-PC was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for LB Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine.

Automatic diagnosis of premature ventricular contraction based on Lyapunov exponents and LVQ neural network.

Premature ventricular contraction (PVC) is a common type of abnormal heartbeat. Without early diagnosis and proper treatment, PVC may result in serious harms. Diagnosis of PVC is of great importance in goal-directed treatment and preoperation prognosis. This paper proposes a novel diagnostic method for PVC based on Lyapunov exponents of electrocardiogram (ECG) beats. The methodology consists of preprocessing, feature extraction and classification integrated into the system. PVC beats can be classified and differentiated from other types of abnormal heartbeats by analyzing Lyapunov exponents and training a learning vector quantization (LVQ) neural network. Our algorithm can obtain a good diagnostic result with little features by using single lead ECG data. The sensitivity, positive predictability, and the overall accuracy of the automatic diagnosis of PVC is 90.26%, 92.31%, and 98.90%, respectively. The effectiveness of the new method is validated through extensive tests using data from MIT-BIH database. The experimental results show that the proposed method is efficient and robust.

Critical role of peroxiredoxin 6 in the repair of peroxidized cell membranes following oxidative stress.

Phospholipids are a major structural component of all cell membranes; their peroxidation represents a severe threat to cellular integrity and their repair is important to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A(2) (PLA(2)) activity, plays a critical role in antioxidant defense of the lung and other organs. We investigated the role of Prdx6 in the repair of peroxidized cell membranes in pulmonary microvascular endothelial cells (PMVEC) and isolated mouse lungs treated with tert-butyl hydroperoxide and lungs from mice exposed to hyperoxia (100% O(2)). Lipid peroxidation was evaluated by measurement of thiobarbituric acid reactive substances, oxidation of diphenyl-1-pyrenylphosphine, or ferrous xylenol orange assay. The exposure dose was varied to give a similar degree of lipid peroxidation at the end of exposure in the different models. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild-type PMVEC and perfused lungs but were unchanged in Pxdx6 null preparations. An intermediate degree of repair was observed with PMVEC and lungs that expressed only C47S or D140A mutant Prdx6; the former mutant does not have peroxidase activity, while the latter loses its PLA(2) activity. Prdx6 null mice showed markedly delayed recovery from lipid peroxidation during 20 h observation following exposure to hyperoxia. Thus, Prdx6 plays a critical role in the repair of peroxidized phospholipids in cell membranes and the recovery of lung cells from peroxidative stress; the peroxidase and PLA(2) activity each contribute to the recovery process.

Solution structure of the reduced form of human peroxiredoxin-6 elucidated using zero-length chemical cross-linking and homology modelling.

Peroxiredoxin-6 (PRDX6) is an unusual member of the peroxiredoxin family of antioxidant enzymes that has only one evolutionarily conserved cysteine. It reduces oxidized lipids and reactive oxygen species (ROS) by oxidation of the active-site cysteine (Cys(47)) to a sulfenic acid, but the mechanism for conversion back to a thiol is not completely understood. Moreover, it has phospholipase A2 (PLA2) activity in addition to its peroxidase activity. Interestingly, some biochemical data are inconsistent with a known high-resolution crystal structure of the catalytic intermediate of the protein, and biophysical data indicate that the protein undergoes conformational changes that affect enzyme activity. In order to further elucidate the solution structure of this important enzyme, we used chemical cross-linking coupled with high-resolution MS (CX-MS), with an emphasis on zero-length cross-links. Distance constraints from high confidence cross-links were used in homology modelling experiments to determine a solution structure of the reduced form of the protein. This structure was further evaluated using chemical cross-links produced by several homo-bifunctional amine-reactive cross-linking reagents, which helped to confirm the solution structure. The results show that several regions of the reduced version of human PRDX6 are in a substantially different conformation from that shown for the crystal structure of the peroxidase catalytic intermediate. The differences between these two structures are likely to reflect catalysis-related conformational changes. These studies also demonstrate that CX-MS using zero-length cross-linking is a powerful strategy for probing protein conformational changes that is complementary to alternative methods such as crystallographic, NMR and biophysical studies.