We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

␣9␣10 nicotinic acetylcholine receptors (nAChRs) have been identified in a variety of tissues including lymphocytes and dorsal root ganglia; except in the case of the auditory system, the function of ␣9␣10 nAChRs is not known. Here we show that selective block (rather than stimulation) of ␣9␣10 nAChRs is analgesic in an animal model of nerve injury pain. In addition, blockade of this nAChR subtype reduces the number of choline acetyltransferase-positive cells, macrophages, and lymphocytes at the site of injury. Chronic neuropathic pain is estimated to affect up to 8% of the world's population; the numerous analgesic compounds currently available are largely ineffective and act through a small number of pharmacological mechanisms. Our findings not only suggest a molecular mechanism for the treatment of neuropathic pain but also demonstrate the involvement of ␣9␣10 nAChRs in the pathophysiology of peripheral nerve injury.N europathic pain is a prolonged, debilitating state characterized by allodynia (pain produced by previously innocuous stimuli), hyperalgesia (an increased or exaggerated response to painful stimuli), and spontaneous pain. Neuropathic pain is often refractory to conventional pain therapeutics such as opioids and nonsteroidal antiinflammatory agents and, therefore, represents a large, unmet clinical need. Neuropathic pain can be triggered in a variety of ways; injury to a peripheral nerve is one of the most common causes.The involvement of nicotinic acetylcholine receptors (nAChRs) in pain has been suggested by a number of experimental observations, and the administration of nAChR agonists reduces pain-related behaviors in several animal models (1-5). nAChRs are pentameric ligand-gated ion channels composed of ␣ (␣1-␣10) and non-␣ (␤1-␤4, , ␥, and ␦) subunits. The ␣2-␣6 and ␤2-␤4 subunits form heteromeric channels consisting of a combination of ␣ and ␤ subunits (6). Homomeric channels can be formed by ␣7 or ␣9 subunits; the ␣10 subunit will only form functional receptors when it is expressed with the ␣9 subunit (6). Many of the nAChRs show widespread patterns of neuronal and nonneuronal distribution; ␣9 and/or ␣10 subunits have been reported within hair cells of the inner ear (7), sperm (8), dorsal root ganglion neurons (9), skin keratinocytes (10), the pars tuberalis of the pituitary (11), and lymphocytes (12). The function of ␣9␣10 nAChRs in the auditory system has been well characterized (13), but little is known regarding the function of ␣9␣10 nAChRs in other tissues. Here we demonstrate that the highly selective antagonist of ␣9␣10 nAChRs, RgIA, is analgesic and reduces migration of macrophages, lymphocytes, and acetylcholine (ACh)-producing cells into the area of nerve injury. ResultsRgIA Is Antinociceptive. Chronic constriction injury (CCI) produced mechanical hypersensitivity within 7 days of sciatic nerve ligation (Fig. 1). Paw withdrawal thresholds (PWTs) were reduced from 122 Ϯ 5 g to 26 Ϯ 5 g 7 days after CCI. The i.m. administration of the ␣9␣10-selective Conus peptide, RgIA, increased...

We present a general-purpose model for biomolecular simulations at the molecular level that incorporates stochasticity, spatial dependence, and volume exclusion, using diffusing and reacting particles with physical dimensions. To validate the model, we first established the formal relationship between the microscopic model parameters (timestep, move length, and reaction probabilities) and the macroscopic coefficients for diffusion and reaction rate. We then compared simulation results with Smoluchowski theory for diffusion-limited irreversible reactions and the best available approximation for diffusion-influenced reversible reactions. To simulate the volumetric effects of a crowded intracellular environment, we created a virtual cytoplasm composed of a heterogeneous population of particles diffusing at rates appropriate to their size. The particle-size distribution was estimated from the relative abundance, mass, and stoichiometries of protein complexes using an experimentally derived proteome catalog from Escherichia coli K12. Simulated diffusion constants exhibited anomalous behavior as a function of time and crowding. Although significant, the volumetric impact of crowding on diffusion cannot fully account for retarded protein mobility in vivo, suggesting that other biophysical factors are at play. The simulated effect of crowding on barnase-barstar dimerization, an experimentally characterized example of a bimolecular association reaction, reveals a biphasic time course, indicating that crowding exerts different effects over different timescales. These observations illustrate that quantitative realism in biosimulation will depend to some extent on mesoscale phenomena that are not currently well understood.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1–3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13–Mms2 is required for DNA damage repair but not nuclear factor κB (NF-κB) activation, whereas Ubc13–Uev1A is involved in NF-κB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

and oncogenesis [1]. The common denominator of these processes is the initial C-terminal activation of ubiquitin The University of Alberta Edmonton, Alberta T6G 2H7 (Ub) by the activating enzyme (E1) followed by its subsequent transfer to a Ub-conjugating enzyme (E2) as a Canada 3 Department of Biochemistry and R.S. covalent E2-Ub thiolester intermediate. At this point, the mechanism of ubiquitination appears to diverge along McLaughlin Macromolecular Structure Facility either of two lines. In one case, Ub is transferred directly from the E2 to the lysine of a target protein in a reaction The University of Western Ontario London, Ontario N6A 5C1 that is facilitated by a Ub protein ligase (E3). In the other case, Ub is first transferred from the E2 to the active site Canada cysteine of an E3 as an E3-Ub thiolester intermediate, whereupon it is then transferred to the target protein. In either event, degradation of the target by the 26S Summary proteasome is facilitated by the assembly of a multi-Ub chain on the target in which the C terminus of each Ub Background: Ubiquitin-conjugating enzymes (E2s) are is linked to Lys48 (K48) of its neighbor. central enzymes involved in ubiquitin-mediated protein The ability of E2 proteins to orchestrate ubiquitination degradation. During this process, ubiquitin (Ub) and the through their interactions with Ub, E1, E3, and target E2 protein form an unstable E2-Ub thiolester intermediproteins makes them central players of the ubiquitin casate prior to the transfer of ubiquitin to an E3-ligase procade. All E2 proteins consist of a catalytic domain (150 tein and the labeling of a substrate for degradation. A residues) that includes the active site cysteine used to series of complex interactions occur among the target form the E2-Ub thiolester complex. Furthermore, X-ray substrate, ubiquitin, E2, and E3 in order to efficiently crystallographic structures of the catalytic domains from facilitate the transfer of the ubiquitin molecule. However, the E2 proteins Ubc1 (vide infra), Ubc2 [2], Ubc7 [3], and due to the inherent instability of the E2-Ub thiolester, Ubc9 [4] from Saccharomyces cerevisiae have shown the structural details of this complex intermediate are that this region is structurally conserved. Four ␣ helices not known. (␣1-␣4) essentially form one face of the protein, while a 4 strand antiparallel ␤ sheet (␤1-␤4) is found on the Results: A three-dimensional model of the E2-Ub thibackside of the enzyme between helices ␣1 and ␣2 in olester intermediate has been determined for the catathe sequence. The thiolester-forming cysteine is located lytic domain of the E2 protein Ubc1 (Ubc1 ⌬450 ) and ubion a relatively unstructured region (L2) ranging from 20 quitin from S. cerevisiae. The interface of the E2-Ub to 30 residues in length and linking ␤4 and ␣2. Recent intermediate was determined by kinetically monitoring X-ray crystallographic studies have shown that two thiolester formation by 1 H-15 N HSQC spectra by using structurally unrelated E3 proteins, E6AP and cCbl, intercombin...

The covalent attachment of ubiquitin (Ub) to short-lived or damaged proteins is believed to be the signal that initiates their selective degradation. In several cases, it has been shown that the proteolytic signal takes the form of a multi-Ub chain in which successive Ub molecules are linked tandemly at lysine 48 (K-48). Here we show that Ub molecules can be linked together in vivo at two other lysine positions, lysine 29 (K-29) and lysine 63 (K-63). The formation of these alternative linkages is strongly dependent on the presence of the stress-related Ub conjugating enzymes UBC4 and UBC5. Furthermore, expression of Ub carrying a K-63 to arginine 63 substitution in a strain of Saccharomyces cerevisiae that is missing the poly-Ub gene, UBI4, fails to compensate for the stress defects associated with these cells. Taken together, these results suggest that the formation of multi-Ub chains involving K-63 linkages plays an important role in the yeast stress response. In broader terms, these results also suggest that Ub is a versatile signal in which different Ub chain configurations are used for different functions.

The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally shortlived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MATa2 repressor of the yeast Saccharemyces cerevisiae is an extremely short-lived protein. We now demonstrate that a2 is conjugated to ubiquitin in vivo. More than one lysine residue of a2 can bejoined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain.Overexpression of degradation-impaired ubiquitin variants was used to show that at least a ficant fraction of a2 degradation is dependent on its ubiquitination.The temporal control of many cellular processes involves short-lived regulators. Proteins that are either conditionally or constitutively short-lived in vivo include cell cycle regulators such as the cyclins (1) and proteins controlling cell differentiation and embryonic development (2-5).A distinct class of intracellular proteolytic pathways involves the covalent ligation of a 76-residue protein, ubiquitin (Ub), to the E-amino groups of acceptor proteins, which are thereby marked for degradation (6). Ub-protein conjugation is catalyzed by a family of Ub-conjugating enzymes, also called E2 enzymes (7). For many short-lived intracellular proteins, their conjugation to Ub is apparently essential for their degradation (8, 9). At the same time, the only specific examples of naturally short-lived proteins whose degradation appears to involve ubiquitination are the phytochrome photoreceptor of plants (10) and cyclins, a family of cell cycle regulators (11).Our recent studies of short-lived proteins included the cell type-specific transcriptional repressor MATa2 of the yeast Saccharomyces cerevisiae (3). This eukaryote has two haploid cell types, a and a, which can mate to form an a/a diploid (12). Ultimately, cell identity is determined by the matingtype, or MAT, locus. In homothallic S. cerevisiae strains, unexpressed a or a information at two other genomic sites is copied into the MAT locus during a mating-type switch.Interconversion between a and a cell types can occur in <90 min. Selective protein degradation might underlie the matingtype switch by causing rapid disappearance of the regulatory proteins involved once their synthesis has ceased.Indeed, the a2 repressor, encoded by the MATa locus and involved in the repression of a cell-specific genes in a cells and of haploid cell-specific genes in a/a diploids, has an in vivo half-life of only -5 min at 30°C (3). Both the aminoterminal and the carboxyl-terminal domains of a2 contain regions that can act as autonomous degradation signals; these signals operate via genetically distinguishable pathways (3).We now demonstrate, using epitope-tagged forms of ubiquitin, that a2 is ubiquitinated in vivo. We also show that ubiquitination of a2 contributes to its rapid degradation.