Bioorthogonal Noncanonical Amino Acid Tagging Research Articles

Exclusive enteral nutrition initiates individual protective microbiome changes to induce remission in pediatric Crohn's disease.

Exclusive enteral nutrition (EEN) is a first-line therapy for pediatric Crohn's disease (CD), but protective mechanisms remain unknown. We established a prospective pediatric cohort to characterize the function of fecal microbiota and metabolite changes of treatment-naive CD patients in response to EEN (German Clinical Trials DRKS00013306). Integrated multi-omics analysis identified network clusters from individually variable microbiome profiles, with Lachnospiraceae and medium-chain fatty acids as protective features. Bioorthogonal non-canonical amino acid tagging selectively identified bacterial species in response to medium-chain fatty acids. Metagenomic analysis identified high strain-level dynamics in response to EEN. Functional changes in diet-exposed fecal microbiota were further validated using gut chemostat cultures and microbiota transfer into germ-free Il10-deficient mice. Dietary model conditions induced individual patient-specific strain signatures to prevent or cause inflammatory bowel disease (IBD)-like inflammation in gnotobiotic mice. Hence, we provide evidence that EEN therapy operates through explicit functional changes of temporally and individually variable microbiome profiles.

CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging

CENCAT enables immunometabolic profiling by measuring protein synthesis via bioorthogonal noncanonical amino acid tagging

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.

Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen

Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life-threatening disease, but their absolute dependence on the host cell has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify effectors delivered into the host cell. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for the genus. The seven novel secreted rickettsial factors (Srfs) we identified include Rickettsia-specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface.

Engineered Proteins and Materials Utilizing Residue-Specific Noncanonical Amino Acid Incorporation.

The incorporation of noncanonical amino acids into proteins and protein-based materials has significantly expanded the repertoire of available protein structures and chemistries. Through residue-specific incorporation, protein properties can be globally modified, resulting in the creation of novel proteins and materials with diverse and tailored characteristics. In this review, we highlight recent advancements in residue-specific incorporation techniques as well as the applications of the engineered proteins and materials. Specifically, we discuss their utility in bio-orthogonal noncanonical amino acid tagging (BONCAT), fluorescent noncanonical amino acid tagging (FUNCAT), threonine-derived noncanonical amino acid tagging (THRONCAT), cross-linking, fluorination, and enzyme engineering. This review underscores the importance of noncanonical amino acid incorporation as a tool for the development of tailored protein properties to meet diverse research and industrial needs.

Multicellular magnetotactic bacteria are genetically heterogeneous consortia with metabolically differentiated cells.

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

Bioorthogonal non-canonical amino acid tagging to track transplanted human induced pluripotent stem cell-specific proteome

BackgroundHuman induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo.MethodsIn this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts.ResultsWe demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs.ConclusionThe novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.

Adaptation to tolerate high doses of arabinoxylan is associated with fecal levels of Bifidobacterium longum

ABSTRACT Dietary fiber supplements are a strategy to close the ‘fiber gap’ and induce targeted modulations of the gut microbiota. However, higher doses of fiber supplements cause gastrointestinal (GI) symptoms that differ among individuals. What determines these inter-individual differences is insufficiently understood. Here we analyzed findings from a six-week randomized controlled trial that evaluated GI symptoms to corn bran arabinoxylan (AX; n = 15) relative to non-fermentable microcrystalline cellulose (MCC; n = 16) at efficacious supplement doses of 25 g/day (females) or 35 g/day (males) in adults with excess weight. Self-reported flatulence, bloating, and stomach aches were evaluated weekly. Bacterial taxa involved in AX fermentation were identified by bioorthogonal non-canonical amino acid tagging. Associations between GI symptoms, fecal microbiota features, and diet history were systematically investigated. AX supplementation increased symptoms during the first three weeks relative to MCC (p < 0.05, Mann-Whitney tests), but subjects ‘adapted’ with symptoms reverting to baseline levels toward the end of treatment. Symptom adaptations were individualized and correlated with the relative abundance of Bifidobacterium longum at baseline (rs = 0.74, p = 0.002), within the bacterial community that utilized AX (rs = 0.69, p = 0.006), and AX-induced shifts in acetate (rs = 0.54, p = 0.039). Lower baseline consumption of animal-based foods and higher whole grains associated with less severity and better adaptation. These findings suggest that humans do ‘adapt’ to tolerate efficacious fiber doses, and this process is linked to their microbiome and dietary factors known to interact with gut microbes, providing a basis for the development of strategies for improved tolerance of dietary fibers.

Diversity of marine bacteria growing on leachates from virgin and weathered plastic: Insights into potential degraders.

Plastic debris in the ocean releases chemical compounds that can be toxic to marine fauna. It was recently found that some marine bacteria can degrade such leachates, but information on the diversity of these bacteria is mostly lacking. In this study, we analysed the bacterial diversity growing on leachates from new low-density polyethylene (LDPE) and a mix of naturally weathered plastic, collected from beach sand. We used a combination of Catalysed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH), BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT), and 16S rRNA gene amplicon sequencing to analyse bacterioplankton-groups specific activity responses and the identity of the responsive taxa to plastic leachates produced under irradiated and non-irradiated conditions. We found that some generalist taxa responded to all leachates, most of them belonging to the Alteromonadales, Oceanospirillales, Nitrosococcales, Rhodobacterales, and Sphingomonadales orders. However, there were also non-generalist taxa responding to specific irradiated and non-irradiated leachates. Our results provide information about bacterial taxa that could be potentially used to degrade the chemicals released during plastic degradation into seawater contributing to its bioremediation.

Plasma growth factors maintain constitutive translation in platelets to regulate reactivity and thrombotic potential

AbstractMechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma–borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography–tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5′-diphosphate (ADP) or thromboxane analog but not convulxin. ADP–induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein–coupled receptor reactivity to balance hemostasis and thrombotic potential.

Biomass distribution and activity of respective subsurface sediments and groundwater within a shallow subsurface ecosystem

Subsurface environments represent diverse microbial communities responsible for mediating biogeochemical cycles linked to the turnover of organic and inorganic carbon important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the different sediment environments, microorganisms typically reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the respective contributions of groundwater (planktonic) and sediment-associated (biofilm) cells to subsurface processes is largely unresolved. In order to directly compare the distribution of microbial biomass and activity in a shallow, subsurface environment, total cell numbers, translationally-active cell numbers (Bioorthogonal non-canonical amino acid tagging- BONCAT), and microbial activity (3H-Leucine incorporation) were investigated for a low biomass pristine and contaminated groundwater and corresponding soil cores. The results demonstrated that cell numbers for the 0.2 um fraction were approximately an order of magnitude higher for the pristine groundwater compared to the contaminated groundwater (106 v. 105). When contaminated groundwater was compared to the pristine, there was a drastic reduction in the BONCAT activity and the contaminated groundwater was between 100-700-fold less. Additionally, the rate of leucine incorporation (3H-leucine) on a per cell basis in pristine groundwater was up to 1,000 times greater than the contaminated groundwater, respectively. Overall, like total cell numbers, activity was lower (both per volume and per cell) in contaminated groundwater compared to pristine groundwater. In pristine soil, activity (3H-leucine) displayed steep gradients of microbial activity in association with transition zones of water table height (i.e., vadose, capillary fringe, saturated). A similar trend was also observed for the contaminated soil; however, the contaminated soil displayed an overall gradient of decreasing activity with depth. The highest activity for pristine soil was 9,253 ng C/g/d located in the transition depth between the capillary fringe and the saturated zone. Conversely, the highest activity for the contaminated soil was 9,175 ng C/g/d located in the vadose zone, perhaps the zone that is least impacted by contaminant flux. The pristine groundwater had higher activity rates than pristine sediment (per cell), but the contaminated groundwater had slower activity rates than the contaminated sediment (per cell). However, for both pristine and contaminated samples on a per volume basis, sediments had the vast majority of microbial activity compared to groundwater (80-95%). In the absence of strong selection forces compared to the contaminated well, the uncontaminated samples demonstrated more phylogenetic differences between the viable and translationally active populations that could be attributed to growth rate differences. The contaminated groundwater sample was predominated by a single, persistent Rhodanobacter strain in the viable fraction, while Rhodococcus, Brevundimonas, and Pseudomonas species dominated the translationally active fraction. Overall, the top active ASVs were prevalent and persistent across the estimated landscape. This is the first quantitative comparison between corresponding groundwater and subsurface sediments as well as predictions of viable and active ASVs (e.g., stable analog probing- SAP) within commonly used sequencing methods. The results suggest that field sampling schemes should consist of both viability and activity-based assessments that can help delineate key microbial populations within diverse microbial communities across and within subsurface systems.

Natural and anthropogenic carbon input affect microbial activity in salt marsh sediment.

Salt marshes are dynamic, highly productive ecosystems positioned at the interface between terrestrial and marine systems. They are exposed to large quantities of both natural and anthropogenic carbon input, and their diverse sediment-hosted microbial communities play key roles in carbon cycling and remineralization. To better understand the effects of natural and anthropogenic carbon on sediment microbial ecology, several sediment cores were collected from Little Sippewissett Salt Marsh (LSSM) on Cape Cod, MA, USA and incubated with either Spartina alterniflora cordgrass or diesel fuel. Resulting shifts in microbial diversity and activity were assessed via bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Both Spartina and diesel amendments resulted in initial decreases of microbial diversity as well as clear, community-wide shifts in metabolic activity. Multi-stage degradative frameworks shaped by fermentation were inferred based on anabolically active lineages. In particular, the metabolically versatile Marinifilaceae were prominent under both treatments, as were the sulfate-reducing Desulfovibrionaceae, which may be attributable to their ability to utilize diverse forms of carbon under nutrient limited conditions. By identifying lineages most directly involved in the early stages of carbon processing, we offer potential targets for indicator species to assess ecosystem health and highlight key players for selective promotion of bioremediation or carbon sequestration pathways.

BONCAT-FACS-Seq reveals the active fraction of a biocrust community undergoing a wet-up event.

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event.

Intercellular contacts affect secretion and biosynthesis of pancreatic islet cells.

Cell protein biosynthesis is regulated by different factors, but implication of intercellular contacts on alpha and beta cell protein biosyntheses activity has not been yet investigated. Islet cell biosynthetic activity is essential in regulating not only the hormonal reserve within cells but also in renewing all the proteins involved in the control of secretion. Here we aimed to assess whether intercellular interactions affected similarly secretion and protein biosynthesis of rat alpha and beta cells. Insulin and glucagon secretion were analyzed by ELISA or reverse hemolytic plaque assay, and protein biosynthesis evaluated at single cell level using bioorthogonal noncanonical amino acid tagging. Regarding beta cells, we showed a positive correlation between insulin secretion and protein biosynthesis. We also observed that homologous contacts increased both activities at low or moderate glucose concentrations. By contrast, at high glucose concentration, homologous contacts increased insulin secretion and not protein biosynthesis. In addition, heterogeneous contacts between beta and alpha cells had no impact on insulin secretion and protein biosynthesis. Regarding alpha cells, we showed that when they were in contact with beta cells, they increased their glucagon secretion in response to a drop of glucose concentration, but, on the other hand, they decreased their protein biosynthesis under any glucose concentrations. Altogether, these results emphasize the role of intercellular contacts on the function of islet cells, showing that intercellular contacts increased protein biosynthesis in beta cells, except at high glucose, and decreased protein biosynthesis in alpha cells even when glucagon secretion is stimulated.

Optimized Bioorthogonal Non-canonical Amino Acid Tagging to Identify Serotype-Specific Biomarkers in Verotoxigenic Escherichia coli.

According to Canada's Food Report Card 2016, there are 4 million foodborne illnesses acquired each year in the nation alone. The leading causes of foodborne illness are pathogenic bacteria such as shigatoxigenic/verotoxigenic Escherichia coli (STEC/VTEC) and Listeria monocytogenes. Most current detection methods used to identify these bacterial pathogens are limited in their validity since they are not specific to detecting metabolically active organisms, potentially generating false-positive results from non-living or non-viable bacteria. Previously, our lab developed an optimized bioorthogonal non-canonical amino acid tagging (BONCAT) method which allows for the labeling of translationally active wild-type pathogenic bacteria. Incorporation of homopropargyl glycine (HPG) into the cellular surfaces of bacteria allows for protein tagging using the bioorthogonal alkyne handle to report on the presence of pathogenic bacteria. Here, we use proteomics to identify more than 400 proteins differentially detected by BONCAT between at least two of five different VTEC serotypes. These findings pave the way for future examination of these proteins as biomarkers in BONCAT-utilizing assays.

Translatome analyses by bio-orthogonal non-canonical amino acid labeling reveal that MR1-activated MAIT cells induce an M1 phenotype and antiviral programming in antigen-presenting monocytes.

MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1β, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

In-Depth Proteomic Analysis of De Novo Proteome in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common dementia syndrome in the elderly characterized by synaptic failure and unique brain pathology. De novo protein synthesis is required for the maintenance of memory and synaptic plasticity. Mounting evidence links impaired neuronal protein synthesis capacity and overall protein synthesis deficits to AD pathogenesis. Meanwhile, identities of AD-associated dysregulation of "newly synthesized proteome" remain elusive. To investigate de novo proteome alterations in the hippocampus of aged Tg19959 AD model mice. In this study, we combined the bioorthogonal noncanonical amino acid tagging (BONCAT) method with the unbiased large-scale proteomic analysis in acute living brain slices (we name it "BONSPEC") to investigate de novo proteome alterations in the hippocampus of Tg19959 AD model mice. We further applied multiple bioinformatics methods to analyze in-depth the proteomics data. In total, 1,742 proteins were detected across the 10 samples with the BONSPEC method. After exclusion of those only detected in less than half of the samples in both groups, 1,362 proteins were kept for further analysis. 37 proteins were differentially expressed (based on statistical analysis) between the WT and Tg19959 groups. Among them, 19 proteins were significantly decreased while 18 proteins were significantly increased in the hippocampi of Tg19959 mice compared to WT mice. The results suggest that proteins involved in synaptic function were enriched in de novo proteome of AD mice. Our study could provide insights into the future investigation into the molecular signaling mechanisms underlying AD and related dementias (ADRDs).

Regulators of proteostasis are translationally repressed in fibroblasts from patients with sporadic and LRRK2-G2019S Parkinson’s disease

Deficits in protein synthesis are associated with Parkinson’s disease (PD). However, it is not known which proteins are affected or if there are synthesis differences between patients with sporadic and Leucine-Rich Repeat Kinase 2 (LRRK2) G2019S PD, the most common monogenic form. Here we used bio-orthogonal non-canonical amino acid tagging for global analysis of newly translated proteins in fibroblasts from sporadic and LRKK2-G2019S patients. Quantitative proteomic analysis revealed that several nascent proteins were reduced in PD samples compared to healthy without any significant change in mRNA levels. Using targeted proteomics, we validated which of these proteins remained dysregulated at the static proteome level and found that regulators of endo-lysosomal sorting, mRNA processing and components of the translation machinery remained low. These proteins included autophagy-related protein 9A (ATG9A) and translational stability regulator YTH N6-ethyladenosine RNA binding protein 3 (YTHDF3). Notably, 77% of the affected proteins in sporadic patients were also repressed in LRRK2-G2019S patients (False discovery rate (FDR) < 0.05) in both sporadic and LRRK2-G2019S samples. This analysis of nascent proteomes from PD patient skin cells reveals that regulators of proteostasis are repressed in both sporadic and LRRK2-G2019S PD.

Differential toxicity of bioorthogonal non-canonical amino acids (BONCAT) in Escherichia coli

Single-cell methods allow studying the activity of single bacterial cells, potentially shedding light on regulatory mechanisms involved in services like biochemical cycling. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising method for studying bacterial activity in natural communities, using the methionine analogues L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG) to track protein production in single cells. Both AHA and HPG have been deemed non-toxic, but recent findings suggest that HPG affects bacterial metabolism. In this study we examined the effect of AHA and HPG on Escherichia coli with respect to acute toxicity and growth. E. coli exposed to 5.6–90 μM HPG showed no growth, and the growth rate was significantly reduced at >0.35 μM HPG, compared to the HPG-free control. In contrast, E. coli showed growth at concentrations up to 9 mM AHA. In assays where AHA or HPG were added during the exponential growth phase, the growth sustained but the growth rate was immediately reduced at the highest concentrations (90 μM HPG and 10 mM AHA). Prolonged incubations (20h) with apparently non-toxic concentrations suggest that the cells incorporating NCAAs fail to divide and do not contribute to the next generation resulting in the relative abundance of labelled cells to decrease over time. These results show that HPG and AHA have different impact on the growth of E. coli. Both concentration and incubation time affect the results and need to be considered when designing BONCAT experiments and evaluating results. Time course incubations are suggested as a possible way to obtain more reliable results.

BONCAT-based Profiling of Nascent Small and Alternative Open Reading Frame-encoded Proteins.

RIBO-seq and proteogenomics have revealed that mammalian genomes harbor thousands of unannotated small and alternative open reading frames (smORFs, <100 amino acids, and alt-ORFs, >100 amino acids, respectively). Several dozen mammalian smORF-encoded proteins (SEPs) and alt-ORF-encoded proteins (alt-proteins) have been shown to play important biological roles, while the overwhelming majority of smORFs and alt-ORFs remain uncharacterized, particularly at the molecular level. Functional proteomics has the potential to reveal key properties of unannotated SEPs and alt-proteins in high throughput, and an approach to identify SEPs and alt-proteins undergoing regulated synthesis should be of broad utility. Here, we introduce a chemoproteomic pipeline based on bio-orthogonal non-canonical amino acid tagging (BONCAT) (Dieterich et al., 2006) to profile nascent SEPs and alt-proteins in human cells. This approach is able to identify cellular stress-induced and cell-cycle regulated SEPs and alt-proteins in cells. Graphical abstract Schematic overview of BONCAT-based chemoproteomic profiling of nascent, unannotated small and alternative open reading frame-encoded proteins (SEPs and alt-proteins).