A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

In Situ Reconstitution of the Adenosine A2A Receptor in Spontaneously Formed Synthetic Liposomes.

Cell transmembrane receptors play a key role in the detection of environmental stimuli and control of intracellular communication. G protein-coupled receptors constitute the largest transmembrane protein family involved in cell signaling. However, current methods for their functional reconstitution in biomimetic membranes remain both challenging and limited in scope. Herein, we describe the spontaneous reconstitution of adenosine A2A receptor (A2AR) during the de novo formation of synthetic liposomes via native chemical ligation. The approach takes advantage of a nonenzymatic and chemoselective method to rapidly generate A2AR embedded phospholiposomes from receptor solubilized in n-dodecyl-ß-d-maltoside analogs. In situ lipid synthesis for protein reconstitution technology proceeds in the absence of dialysis and/or detergent absorbents, and A2AR assimilation into synthetic liposomes can be visualized by microscopy and probed by radio-ligand binding.

Encapsulation of Living Cells within Giant Phospholipid Liposomes Formed by the Inverse-Emulsion Technique.

Liposomes form spontaneously by the assimilation of phospholipids, the primary component of cell membranes. Due to their unique ability to form selectively permeable bilayers in situ, they are widely used as nanocarriers for drug and small-molecule delivery. However, there is a lack of straightforward methodologies to encapsulate living microorganisms. Here we demonstrate the successful encapsulation of whole cells in phospholipid vesicles by using the inverse-emulsion technique of generating unilamellar vesicles. This method of liposome preparation allows for a facile encapsulation of large biomaterials that previously was not easily attainable. Using Escherichia coli as a model organism, we found that liposomes can protect the bacterium against external protease degradation and from harsh biological environments. Liposomes prepared by the inverse-emulsion method were also capable of encapsulating yeast and were found to be naturally susceptible to hydrolysis by enzymes such as phospholipases, thus highlighting their potential role as cell delivery carriers.

Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation.

We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a polyethylene glycol linker attached to the exocyclic amine of PreQ1. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleobases using a simple hairpin recognition element.

Spontaneous Reconstitution of Functional Transmembrane Proteins During Bioorthogonal Phospholipid Membrane Synthesis.

Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes in situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes.

Self-reproducing catalyst drives repeated phospholipid synthesis and membrane growth.

Cell membranes are dynamic structures found in all living organisms. There have been numerous constructs that model phospholipid membranes. However, unlike natural membranes, these biomimetic systems cannot sustain growth owing to an inability to replenish phospholipid-synthesizing catalysts. Here we report on the design and synthesis of artificial membranes embedded with synthetic, self-reproducing catalysts capable of perpetuating phospholipid bilayer formation. Replacing the complex biochemical pathways used in nature with an autocatalyst that also drives lipid synthesis leads to the continual formation of triazole phospholipids and membrane-bound oligotriazole catalysts from simpler starting materials. In addition to continual phospholipid synthesis and vesicle growth, the synthetic membranes are capable of remodeling their physical composition in response to changes in the environment by preferentially incorporating specific precursors. These results demonstrate that complex membranes capable of indefinite self-synthesis can emerge when supplied with simpler chemical building blocks.

Bioorthogonal tetrazine-mediated transfer reactions facilitate reaction turnover in nucleic acid-templated detection of microRNA.

Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates.

In situ vesicle formation by native chemical ligation.

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non-enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water-soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self-assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.

Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives.

Sugar coated: We recently developed methylcyclopropenes as low-molecular-weight tetrazine coupling partners. Here, we demonstrate that methylcyclopropenes can meet the stringent steric demands required for metabolic imaging of unnatural mannosamines on live cells. Using sequential azide-alkyne chemistry, we also demonstrate multicolor imaging of two different metabolically incorporated unnatural sugars.