Repurposed 3D Printer Allows Economical and Programmable Fraction Collection for Proteomics of Nanogram Scale Samples.

In this work, we describe the construction and application of a repurposed 3D-printer as a fraction collector. We utilize a nano-LC to ensure minimal volumes and surfaces although any LC can be coupled. The setup operates as a high-pH fractionation system capable of effectively working with nanogram scales of lysate digests. The 2D RP-RP system demonstrated superior proteome coverage over single-shot data-dependent acquisition (DDA) analysis using only 5 ng of human cell lysate digest with performance increasing with increasing amounts of material. We found that the fractionation system allowed over 60% signal recovery at the peptide level and, more importantly, we observed improved protein level intensity coverage, which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of data-independent acquisition (DIA) and wide window acquisition (WWA) to fractionated samples allowed nearly 8000 proteins to be identified from 50 ng of the material. The utility of the 2D system was further investigated for phosphoproteomics (>21 000 phosphosites from 50 µg starting material) and pull-down type experiments and showed substantial improvements over single-shot experiments. We show that the 2D RP-RP system is a highly versatile and powerful tool for many proteomics workflows.

Direct radical functionalization of native sugars.

Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free 'cap and glycosylate' approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.

The Minimum Protein Staple? - Towards 'bio'-Baldwin's rules via inter-phosphosite linking in the MEK1 activation loop.

In small molecule organic chemistry, the heuristic insight into ring-forming processes that was enabled by Baldwin's rules some 50 years ago proved a step-change in the role of mechanistically guided synthesis. It created a lens upon and marker of fundamental stereoelectronic and conformation-guided chemical processes. However, despite the widespread role of stereoelectronics and conformational control in Biology, no equivalent coherent exploitation of trapped, ring-forming processes yet exists in biomolecules. In the development of a minimal ring-closing process in intact proteins that might prove suitable in a coherent rule-set, we have tested endo-trig ring-closing conjugate thioether lanthionine (Lan) -CH2-S-CH2- formation as a limiting cyclization. Spontaneous Lan formation in proteins is rare if not non-existent and when found in natural product cyclic peptides it requires the mediation of corresponding biosynthetic enzymes as well as productive reactive conformations to guide it. Here, we show that within a conformationally flexible and functionally important protein loop - the MAPK kinase phosphorylation-targeted activation loop - Lan ring-closing is possible. Ring-closing proves to be critically dependent on the location of a trig electrophilic site in just one of two regioisomeric potential precursors to allow phosphosite-to-phosphosite 'stapling'. This first example of spontaneous protein thioether ring-closing/'stapling' and its accessibility from just one precursor (despite the potential for both to form an identical 'staple') now reveals the potential for Lan formation not only as an accessible form of minimal stapling in proteins but also as an exquisitely sensitive probe of associated protein geometries. We suggest that the use of this (as well as the development of other such, intramolecular protein traps that are dependent on inherent protein-controlled reactivity rather than forced crosslinking) may allow the broader trapping and mapping of relevant, even minor, protein states. In this way, protein ring formation may enable a form of extended 'bio-Baldwin's rules' that help to delineate relevant protein conformational space.

Characterization of an Omnitrap-Orbitrap Platform Equipped with Infrared Multiphoton Dissociation, Ultraviolet Photodissociation, and Electron Capture Dissociation for the Analysis of Peptides and Proteins.

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions that can be used to perform resonant-based collision-induced dissociation, several forms of electron-associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation in a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the investigation of polypeptides.

Stereoretentive Post-Translational Protein Editing.

Chemical post-translational methods allow convergent side-chain editing of proteins without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side chains via C-C bond formation, using off-protein carbon-centered C· radicals added to unnatural amino acid radical acceptor (SOMOphile, singly occupied molecular orbital (SOMO)) "tags" such as dehydroalanine, are benign and wide-ranging. However, they also typically create epimeric mixtures of d/l-residues. Here, we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-proteinl-alanyl Cß· radicals, allows Cß-Hγ, Cß-Oγ, Cß-Seγ, Cß-Bγ, and Cß-Cγ bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid precursor. This methodology shows great potential to explore protein side-chain diversity and function and in the construction of useful bioconjugates.

Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy.

BACKGROUND: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac ß-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. METHODS: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with ß-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. RESULTS: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. CONCLUSIONS: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

Functional Outcomes and Successful Predictors of Lumbar Transforaminal Epidural Steroid Injections (LTFESIs) for Lumbar Radiculopathy Under Fluoroscopic Guidance: A Prospective Study.

BACKGROUND: Lumbar radiculopathy, a common and debilitating condition, often necessitates a multimodal approach for effective management. Lumbar transforaminal epidural steroid injection (LTFESI) has emerged as a valuable therapeutic option when conservative measures fall short. Recent interest in long-acting and non-particulate steroids prompts a critical examination of their impact on LTFESI outcomes. This prospective study aims to evaluate the efficacy of LTFESI in improving pain and functional outcomes in patients with lumbar radiculopathy, focusing on long-acting and non-particulate steroids, and analyse the associated economic burden. METHODS: The study, conducted from October 2017 to April 2019, involved 52 patients with lumbar radiculopathy meeting specific criteria. LTFESI was administered using a hospital-based prospective design. Functional outcomes were assessed using the Oswestry Disability Index (ODI) and Numeric Rating Scale (NRS) scores at various intervals. Statistical analyses were performed to identify predictors of successful outcomes. RESULTS: Participants (average age 43.22 years, 27 (51.92%) male) exhibited diverse Michigan State University (MSU) grade profiles and predominantly had pathology at the L4-5 level. The study demonstrated a significant and lasting functional improvement in 43 (82.69%) of patients after LTFESI. Patients with 2AB-type intervertebral disc prolapse (IVDP) showed lower response rates, emphasizing subtype influence. The efficacy of LTFESI was sustained for up to six months in almost 82.69% of patients, highlighting its potential for long-lasting benefits. The difference in the mean ODI score pre-injection and six months post-injection is statistically significant (p<0.0001). A total of four patients (7.69%) underwent surgical treatment for lumbar radiculopathy as their symptoms did not improve after injection. For all four patients (7.69%), surgery was done one month after injection. Five patients (9.61%) had ODI scores of more than 40, indicating severe disability at the end of six months. So, in nine patients (17.3%), the injection given was not effective at the end of six months, four (7.69%) of whom were operated on and five (9.61%) patients received conservative treatment. Thus, 43 (82.69%) of patients had a good outcome. DISCUSSION: The study reinforces LTFESI as an effective and safe intervention, providing substantial and lasting benefits for lumbar radiculopathy. The majority experienced immediate relief, supporting its role as an intermediate option between conservative management and surgery. Identified predictors of decreased success underscore the importance of early intervention and tailored treatment plans. The study emphasizes LTFESI's diagnostic and therapeutic potential, with economic benefits and safety highlighted. CONCLUSION: LTFESI emerges as a safe and effective intervention for lumbar radiculopathy, offering substantial and enduring pain relief. The study contributes valuable insights into the nuanced outcomes of LTFESI, including the impact of IVDP subtypes, factors influencing success, and the procedure's cost-effectiveness. While acknowledging limitations, this work adds to the growing evidence supporting LTFESI as a crucial component in the management of lumbar radiculopathy.

Lipids mediate supramolecular outer membrane protein assembly in bacteria.

ß Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric ß barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis.

Photosynthesis is the energetic basis for most life on Earth, and in plants it operates inside double membrane-bound organelles called chloroplasts. The photosynthetic apparatus comprises numerous proteins encoded by the nuclear and organellar genomes. Maintenance of this apparatus requires the action of internal chloroplast proteases, but a role for the nucleocytosolic ubiquitin-proteasome system (UPS) was not expected, owing to the barrier presented by the double-membrane envelope. Here, we show that photosynthesis proteins (including those encoded internally by chloroplast genes) are ubiquitinated and processed via the CHLORAD pathway: They are degraded by the 26S proteasome following CDC48-dependent retrotranslocation to the cytosol. This demonstrates that the reach of the UPS extends to the interior of endosymbiotically derived chloroplasts, where it acts to regulate photosynthesis, arguably the most fundamental process of life.

Broad-range metalloprotease profiling in plants uncovers immunity provided by defence-related metalloenzyme.

Plants encode > 100 metalloproteases representing > 19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant research. We describe the use of hydroxamate-based photoaffinity probes to explore plant proteomes for metalloproteases. We detected labelling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to nine different metalloprotease families and localize to different subcellular compartments. The probes identified several chloroplastic FtsH proteases, vacuolar aspartyl aminopeptidase DAP1, peroxisomal metalloprotease PMX16, extracellular matrix metalloproteases and many cytosolic metalloproteases. We also identified nonproteolytic metallohydrolases involved in the release of auxin and in the urea cycle. Studies on tobacco plants (Nicotiana benthamiana) infected with the bacterial plant pathogen Pseudomonas syringae uncovered the induced labelling of PRp27, a secreted protein with implicated metalloprotease activity. PRp27 overexpression increases resistance, and PRp27 mutants lacking metal binding site are no longer labelled, but still show increased immunity. Collectively, these studies reveal the power of broad-range metalloprotease profiling in plants using hydroxamate-based probes.

Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens.

Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (H2O2) identified ~200 H2O2-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these H2O2-sensitive Cys residues are more reactive in response to H2O2 and several proteins have multiple Cys residues with opposite reactivities in response to H2O2 exposure.

Reductive site-selective atypical C,Z-type/N2-C2 cleavage allows C-terminal protein amidation.

Biomolecule environments can enhance chemistries with the potential to mediate and modulate self-modification (e.g., self-cleavage). While these enhanced modes are found in certain biomolecules (e.g., RNA ribozymes), it is more rare in proteins. Targeted proteolytic cleavage is vital to physiology, biotechnology, and even emerging therapy. Yet, purely chemically induced methods for the site-selective cleavage of proteins remain scarce. Here, as a proof of principle, we designed and tested a system intended to combine protein-enhanced chemistry with tag modification to enable synthetic reductive protein chemistries promoted by diboron. This reductively driven, single-electron chemistry now enables an operationally simple, site-selective cleavage protocol for proteins directed to readily accessible dehydroalanine (Dha) residues as tags under aqueous conditions and in cell lysates. In this way, a mild, efficient, enzyme-free method now allows not only precise chemical proteolysis but also simultaneous use in the removal of affinity tags and/or protein-terminus editing to create altered N- and C-termini such as protein amidation (─CONH2).

Uncovering viral RNA-host cell interactions on a proteome-wide scale.

RNA viruses interact with a wide range of cellular RNA-binding proteins (RBPs) during their life cycle. The prevalence of these host-virus interactions has been highlighted by new methods that elucidate the composition of viral ribonucleoproteins (vRNPs). Applied to 11 viruses so far, these approaches have revealed hundreds of cellular RBPs that interact with viral (v)RNA in infected cells. However, consistency across methods is limited, raising questions about methodological considerations when designing and interpreting these studies. Here, we discuss these caveats and, through comparing available vRNA interactomes, describe RBPs that are consistently identified as vRNP components and outline their potential roles in infection. In summary, these novel approaches have uncovered a new universe of host-virus interactions holding great therapeutic potential.

RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response.

The nuclear RNA exosome plays a key role in controlling the levels of multiple protein-coding and non-coding RNAs. Recruitment of the exosome to specific RNA substrates is mediated by RNA-binding co-factors. The transient interaction between co-factors and the exosome as well as the rapid decay of RNA substrates make identification of exosome co-factors challenging. Here, we use comparative poly(A)+ RNA interactome capture in fission yeast expressing three different mutants of the exosome to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identify multiple RNA-binding proteins whose association with RNA is altered in exosome mutants, including the zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc-finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight the importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress.

Post-translational insertion of boron in proteins to probe and modulate function.

Boron is absent in proteins, yet is a micronutrient. It possesses unique bonding that could expand biological function including modes of Lewis acidity not available to typical elements of life. Here we show that post-translational Cß-Bγ bond formation provides mild, direct, site-selective access to the minimally sized residue boronoalanine (Bal) in proteins. Precise anchoring of boron within complex biomolecular systems allows dative bond-mediated, site-dependent protein Lewis acid-base-pairing (LABP) by Bal. Dynamic protein-LABP creates tunable inter- and intramolecular ligand-host interactions, while reactive protein-LABP reveals reactively accessible sites through migratory boron-to-oxygen Cß-Oγ covalent bond formation. These modes of dative bonding can also generate de novo function, such as control of thermo- and proteolytic stability in a target protein, or observation of transient structural features via chemical exchange. These results indicate that controlled insertion of boron facilitates stability modulation, structure determination, de novo binding activities and redox-responsive 'mutation'.

Colicin-Mediated Transport of DNA through the Iron Transporter FepA.

Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.

Global analysis of protein-RNA interactions in SARS-CoV-2-infected cells reveals key regulators of infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

LanCLs add glutathione to dehydroamino acids generated at phosphorylated sites in the proteome.

Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such "elimination damage" generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically.

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein.

The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.