Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation. It is most commonly known as a selection marker for cell lines genetically engineered to express a resistance transgene, but its additional uses as a probe for protein synthesis have proven invaluable across a wide variety of model systems, ranging from purified ribosomes and cell-free translation to intact cultured cells and whole animals. Puromycin is comprised of a nucleoside covalently bound to an amino acid, mimicking the 3' end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes. Both moieties can tolerate some chemical substitutions and modifications without significant loss of activity, generating a diverse toolbox of puromycin-based reagents with added functionality, such as biotin for affinity purification or fluorophores for fluorescent microscopy detection. These reagents, as well as anti-puromycin antibodies, have played a pivotal role in advancing our understanding of the regulation and dysregulation of protein synthesis in normal and pathological processes, including immune response and neurological function. This manuscript reviews the current state of puromycin-based research, including structure and mechanism of action, relevant derivatives, use in advanced methodologies and some of the major insights generated using such techniques both in the lab and the clinic.

The science of puromycin: From studies of ribosome function to applications in biotechnology

Alzheimer's disease beyond amyloid: Can the repetitive failures of amyloid-targeted therapeutics inform future approaches to dementia drug discovery?

Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~ 3 nm × 4 nm) is ~ 7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on TauFL or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of TauRD filaments.

/pdf/fret-based-tau-seeding-assay-does-not-represent-prion-like-2v6rydv6yt.pdf

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments.

Tau neurofibrillary tangle pathology characterizes Alzheimer’s disease and other neurodegenerative tauopathies. Brain gene expression profiles can reveal mechanisms; however, few studies have systematically examined both the transcriptome and proteome or differentiated Tau- versus age-dependent changes. Paired, longitudinal RNA-sequencing and mass-spectrometry were performed in a Drosophila model of tauopathy, based on pan-neuronal expression of human wildtype Tau (TauWT) or a mutant form causing frontotemporal dementia (TauR406W). Tau-induced, differentially expressed transcripts and proteins were examined cross-sectionally or using linear regression and adjusting for age. Hierarchical clustering was performed to highlight network perturbations, and we examined overlaps with human brain gene expression profiles in tauopathy. TauWT induced 1514 and 213 differentially expressed transcripts and proteins, respectively. TauR406W had a substantially greater impact, causing changes in 5494 transcripts and 697 proteins. There was a ~ 70% overlap between age- and Tau-induced changes and our analyses reveal pervasive bi-directional interactions. Strikingly, 42% of Tau-induced transcripts were discordant in the proteome, showing opposite direction of change. Tau-responsive gene expression networks strongly implicate innate immune activation. Cross-species analyses pinpoint human brain gene perturbations specifically triggered by Tau pathology and/or aging, and further differentiate between disease amplifying and protective changes. Our results comprise a powerful, cross-species functional genomics resource for tauopathy, revealing Tau-mediated disruption of gene expression, including dynamic, age-dependent interactions between the brain transcriptome and proteome.

/pdf/integrated-analysis-of-the-aging-brain-transcriptome-and-322tay2x1z.pdf

Integrated analysis of the aging brain transcriptome and proteome in tauopathy

There is a fundamental gap in understanding the consequences of tau-ribosome interactions. Tau oligomers and filaments hinder protein synthesis in vitro, and they associate strongly with ribosomes in vivo. Here, we investigated the consequences of tau interactions with ribosomes in transgenic mice, in cells, and in human brain tissues to identify tau as a direct modulator of ribosomal selectivity. First, we performed microarrays and nascent proteomics to measure changes in protein synthesis. Using regulatable rTg4510 tau transgenic mice, we determined that tau expression differentially shifts both the transcriptome and the nascent proteome, and that the synthesis of ribosomal proteins is reversibly dependent on tau levels. We further extended these results to human brains and found that tau pathologically interacts with ribosomal protein S6 (rpS6 or S6), a crucial regulator of translation. Consequently, protein synthesis under translational control of rpS6 was reduced under tauopathic conditions in Alzheimer's disease brains. Our data establish tau as a driver of RNA translation selectivity. Moreover, since regulation of protein synthesis is critical for learning and memory, aberrant tau-ribosome interactions in disease could explain the linkage between tauopathies and cognitive impairment.

/pdf/tau-drives-translational-selectivity-by-interacting-with-1wrrnr8yf4.pdf

Tau drives translational selectivity by interacting with ribosomal proteins.

Carbon dot-based biosensors for the detection of communicable and non -communicable diseases.

Chlorpyrifos formulations are the most used in Colombia and other countries for crop protection, and their presence has been proven in many water sources. The application of nanomaterials, including carbon quantum dots (CD), can significantly improve the performance of optical biosensors for quick and accurate detection of these pesticides. In this work, naturally fluorescent and nontoxic CD were conjugated with acetylcholinesterase (AChE) as a bioreceptor, to produce a fluorescent biosensor. This system was modulated with graphene oxide (GO), showing a fluorescence recovery in the presence of the pesticide. The biosensor was evaluated for the detection of pure chlorpyrifos (CPF), profenofos (PF), and a commercial formulation called Lorsban®. A limit of detection (LOD) as low as 0.14 and 2.05 ppb for chlorpyrifos and Lorsban®, respectively, was obtained. Profenofos did not show any recovery with this system at the evaluated concentrations. The system also showed a good selectivity in the presence of proteins (BSA) and other organic substances (Glucose) usually present in drinking water. The specificity was also evaluated against cypermethrin and methomyl commercial formulations showing minimal interference. To the best of our knowledge, this is one of the few works reporting the detection of commercial pesticide formulations with promising results. Graphical Abstract

/pdf/highly-sensitive-fluorescent-biosensor-based-on-2q64h8fu.pdf

Highly Sensitive Fluorescent Biosensor Based on Acetylcholinesterase and Carbon Dots–Graphene Oxide Quenching Test for Analytical and Commercial Organophosphate Pesticide Detection

Juan Pablo Arango

Papers

Tau drives translational selectivity by interacting with ribosomal proteins.

Carbon dot-based biosensors for the detection of communicable and non -communicable diseases.

Highly Sensitive Fluorescent Biosensor Based on Acetylcholinesterase and Carbon Dots–Graphene Oxide Quenching Test for Analytical and Commercial Organophosphate Pesticide Detection