Gut microbial β-glucuronidases influence endobiotic homeostasis and are modulated by diverse therapeutics.

Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial β-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation.

Prioritized mass spectrometry increases the depth, sensitivity and data completeness of single-cell proteomics.

Major aims of single-cell proteomics include increasing the consistency, sensitivity and depth of protein quantification, especially for proteins and modifications of biological interest. Here, to simultaneously advance all these aims, we developed prioritized Single-Cell ProtEomics (pSCoPE). pSCoPE consistently analyzes thousands of prioritized peptides across all single cells (thus increasing data completeness) while maximizing instrument time spent analyzing identifiable peptides, thus increasing proteome depth.

Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight.

Multiplexed single-cell proteomics using SCoPE2.

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification.

Antibiotic Adjuvant Activity Revealed in a Photoaffinity Approach to Determine the Molecular Target of Antipyocyanin Compounds.

Infections with are a looming threat to public health. New treatment strategies are needed to combat this pathogen, for example, by blocking the production of virulence factors like pyocyanin. A photoaffinity analogue of an antipyocyanin compound was developed to interrogate the inhibitor's molecular mechanism of action.

Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2.

Background: Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis.

Results: To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation.

Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate.

Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase 1 (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG).

Digestomics: an emerging strategy for comprehensive analysis of protein catabolism.

When cells mobilize nutrients from protein, they generate a fingerprint of peptide fragments that reflects the net action of proteases and the identities of the affected proteins. Analyzing these mixtures falls into a grey area between proteomics and metabolomics that is poorly served by existing technology. Herein, we describe an emerging digestomics strategy that bridges this gap and allows mixtures of proteolytic fragments to be quantitatively mapped with an amino acid level of resolution.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms.

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules.

Systems-level analysis of mechanisms regulating yeast metabolic flux.

Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states and, therefore, how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures.

Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

Background: Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling.

Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite.

Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite 'acetylome', characterizing 2,876 acetylation sites on 1,146 proteins.

Mitotic Control of Planar Cell Polarity by Polo-like Kinase 1.

During cell division, polarized epithelial cells employ mechanisms to preserve cell polarity and tissue integrity. In dividing cells of the mammalian skin, planar cell polarity (PCP) is maintained through the bulk internalization, equal segregation, and polarized recycling of cortical PCP proteins. The dramatic redistribution of PCP proteins coincides precisely with cell-cycle progression, but the mechanisms coordinating PCP and mitosis are unknown.

Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively.

Impact of the adenoviral E4 Orf3 protein on the activity and posttranslational modification of p53.

Unlabelled: Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J.

ChelomEx: Isotope-assisted discovery of metal chelates in complex media using high-resolution LC-MS.

Chelating agents can control the speciation and reactivity of trace metals in biological, environmental, and laboratory-derived media. A large number of trace metals (including Fe, Cu, Zn, Hg, and others) show characteristic isotopic fingerprints that can be exploited for the discovery of known and unknown organic metal complexes and related chelating ligands in very complex sample matrices using high-resolution liquid chromatography mass spectrometry (LC-MS). However, there is currently no free open-source software available for this purpose.

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins.

RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm.

Mutated in colorectal cancer (MCC) is a novel oncogene in B lymphocytes.

Background: Identification of novel genetic risk factors is imperative for a better understanding of B lymphomagenesis and for the development of novel therapeutic strategies. TRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. The present study aimed to identify novel oncogenes involved in malignant transformation of TRAF3-deficient B cells.

Functional role of autophagy-mediated proteome remodeling in cell survival signaling and innate immunity.

Ras-driven cancer cells upregulate basal autophagy that degrades and recycles intracellular proteins and organelles. Autophagy-mediated proteome degradation provides free amino acids to support metabolism and macromolecular synthesis, which confers a survival advantage in starvation and promotes tumorigenesis. While the degradation of isolated protein substrates by autophagy has been implicated in controlling cellular function, the extent and specificity by which autophagy remodels the cellular proteome and the underlying functional consequences were unknown.

The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements.

A phosphohistidine proteomics strategy based on elucidation of a unique gas-phase phosphopeptide fragmentation mechanism.

Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC-MS/MS.

Transcription-independent functions of an RNA polymerase II subunit, Rpb2, during genome rearrangement in the ciliate, Oxytricha trifallax.

The RNA polymerase II (Pol-II) holoenzyme, responsible for messenger RNA production, typically consists of 10-12 subunits. Our laboratory previously demonstrated that maternally deposited, long, noncoding, template RNAs are essential for programmed genome rearrangements in the ciliate Oxytricha trifallax. Here we show that such RNAs are bidirectionally transcribed and transported to the zygotic nucleus.