Polyunsaturated Fatty Acid-Derived Lipid Mediators as Potential Biomarkers for Leprosy Among Individuals with Asymptomatic Infection.

Intra-household contacts (HCs) of leprosy patients are at increased risk of infection by and about ∼5-10% will develop active disease. A prognostic tool to identify HCs with the greatest risk of progressing to active disease would enhance early leprosy diagnosis and optimize prophylactic intervention. Previous metabolomics studies suggest that host lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are potential biomarkers for leprosy.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines.

On-Chip Acousto Thermal Shift Assay for Rapid and Sensitive Assessment of Protein Thermodynamic Stability.

Thermal shift assays (TSAs) have been extensively used to study thermodynamics of proteins and provide an efficient means to assess protein-ligand binding or protein-protein interactions. However, existing TSAs have limitations, such as being time consuming, labor intensive, or having low sensitivity. Herein, an acousto thermal shift assay (ATSA), the first ultrasound enabled TSA, is reported for real-time analysis of protein thermodynamic stability.

An isothermal shift assay for proteome scale drug-target identification.

Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput.

A pilot metabolomics study of tuberculosis immune reconstitution inflammatory syndrome.

Background: Diagnosis of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is challenging and new tools are needed for early diagnosis as well as to understand the biochemical events that underlie the pathology in TB-IRIS.

Methods: Plasma samples were obtained from participants from a randomized HIV/TB treatment strategy study (AIDS Clinical Trials Group [ACTG] A5221) with (n = 26) and without TB-IRIS (n = 22) for an untargeted metabolomics pilot study by liquid-chromatography mass spectrometry. The metabolic profile of these participants was compared at the study entry and as close to the diagnosis of TB-IRIS as possible (TB-IRIS window).

Elucidation of a Human Urine Metabolite as a Seryl-Leucine Glycopeptide and as a Biomarker of Effective Anti-Tuberculosis Therapy.

The evaluation of new tuberculosis (TB) therapies is limited by the paucity of biomarkers to monitor treatment response. Previous work detected an uncharacterized urine metabolite with a molecular mass of 874.3547 Da that showed promise as a biomarker for successful TB treatment.

Ubiquitin-dependent switch during assembly of the proteasomal ATPases mediated by Not4 ubiquitin ligase.

In the proteasome holoenzyme, the hexameric ATPases (Rpt1-Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early-stage proteasome assembly, individual Rpt proteins assemble into the hexameric "Rpt ring" through binding to their cognate chaperones: Nas2, Hsm3, Nas6, and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination-mediated control.

Identification of Urine Metabolites as Biomarkers of Early Lyme Disease.

Metabolites detectible in human biofluids are attractive biomarkers for the diagnosis of early Lyme disease (ELD), a vector-borne infectious disease. Urine represents an easily obtained clinical sample that can be applied for diagnostic purposes. However, few studies have explored urine for biomarkers of ELD.

Decoys Untangle Complicated Redundancy and Reveal Targets of Circadian Clock F-Box Proteins.

Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis ().

Dengue viruses cleave STING in humans but not in nonhuman primates, their presumed natural reservoir.

Human dengue viruses emerged from primate reservoirs, yet paradoxically dengue does not reach high titers in primate models. This presents a unique opportunity to examine the genetics of spillover versus reservoir hosts. The dengue virus 2 (DENV2) - encoded protease cleaves human STING, reducing type I interferon production and boosting viral titers in humans.

Metabolic differentiation of early Lyme disease from southern tick-associated rash illness (STARI).

Lyme disease, the most commonly reported vector-borne disease in the United States, results from infection with Early clinical diagnosis of this disease is largely based on the presence of an erythematous skin lesion for individuals in high-risk regions. This, however, can be confused with other illnesses including southern tick-associated rash illness (STARI), an illness that lacks a defined etiological agent or laboratory diagnostic test, and is coprevalent with Lyme disease in portions of the eastern United States. By applying an unbiased metabolomics approach with sera retrospectively obtained from well-characterized patients, we defined biochemical and diagnostic differences between early Lyme disease and STARI.

Type 1 Reaction in Patients With Leprosy Corresponds to a Decrease in Proresolving Lipid Mediators and an Increase in Proinflammatory Lipid Mediators.

Background: Type 1 reaction (T1R) is an acute T-helper type 1 (Th1) inflammatory episode in patients with leprosy. While immunological responses associated with T1R have been investigated, the corresponding metabolic responses that could contribute to T1R pathology have received little attention.

Methods: Metabolomics-based analyses of sera from 7 patients with and 9 without T1R were conducted via liquid chromatography-mass spectrometry.

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase.

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction.

Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction.

Recruitment of the 4EHP-GYF2 cap-binding complex to tetraproline motifs of tristetraprolin promotes repression and degradation of mRNAs with AU-rich elements.

The zinc finger protein tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). Although much attention has been directed toward understanding the decay process and machinery involved, the translation repression role of TTP has remained poorly understood. Here we identify the cap-binding translation repression 4EHP-GYF2 complex as a cofactor of TTP.

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood.

Multimodal mechanism of action for the Cdc34 acidic loop: a case study for why ubiquitin-conjugating enzymes have loops and tails.

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction.

Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast.

A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near-complete yeast proteome from a whole cell tryptic digest. This modified online two-dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient.

Digestion and depletion of abundant proteins improves proteomic coverage.

Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics.

Identification of methylated proteins in the yeast small ribosomal subunit: a role for SPOUT methyltransferases in protein arginine methylation.

We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses.