Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation.

Prions are proteins that can assume at least two distinct conformational states, one of which is dominant and self-perpetuating. Previously we found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy can display prion-like properties in yeast. Here we find that, when exogenously expressed in sensory neurons, ApCPEB can form an amyloidogenic self-sustaining multimer, consistent with it being a prion-like protein. In addition, we find that conversion of both the exogenous and the endogenous ApCPEB to the multimeric state is enhanced by the neurotransmitter serotonin and that an antibody that recognizes preferentially the multimeric ApCPEB blocks persistence of synaptic facilitation. These results are consistent with the idea that ApCPEB can act as a self-sustaining prion-like protein in the nervous system and thereby might allow the activity-dependent change in synaptic efficacy to persist for long periods of time.

mRNA and tRNA modification states influence ribosome speed and frame maintenance during poly(lysine) peptide synthesis.

Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNALys or Val-tRNALys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.

Crosslinked Layered Surfaces of Heparin and Poly(L-Lysine) Enhance Mesenchymal Stromal Cell Behavior in the Presence of Soluble Interferon Gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.

Crystal structure of the adenylation domain from an ε-poly-l-lysine synthetase provides molecular mechanism for substrate specificity.

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain. From the crystal structures of the adenylation domain from NRPSs, the amino acid residues involved in substrate binding can be assumed; however, the precise interactions for better understanding the Pls recognition of l-Lys and its analogs have not yet been fully elucidated. Here, we determined the crystal structure of the adenylation domain of Pls in complex with the intermediate lysyl adenylate at 2.3 Å resolution. This is the first structure determination of the l-Lys activating adenylation domain. The crystal structure reveals that the shape of the substrate-binding pocket determines the specific recognition of l-Lys and its analogs and the electrostatic and hydrogen-bonding interactions further strengthen substrate binding. This study helps us understand the ε-PL synthesis mechanism and contributes to improving our knowledge of the molecular mechanism of NRPS adenylation domains towards their successful application in bioengineering.

Transcriptome and Metabolome Analysis Revealing the Improved ε-Poly-l-Lysine Production Induced by a Microbial Call from Botrytis cinerea.

ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.

Transcriptomic and metabolomic analyses for providing insights into the influence of polylysine synthetase on the metabolism of Streptomyces albulus.

ε-poly-L-lysine (ε-PL) is the main secondary metabolite of Streptomyces albulus, and it is widely used in the food industry. Polylysine synthetase (Pls) is the last enzyme in the ε-PL biosynthetic pathway. Our previous study revealed that Pls overexpressed in S. albulus CICC11022 result in the efficient production of ε-PL. In this study, a Pls gene knockout strain was initially constructed. Then, genomic, transcriptomic and metabolomic approaches were integrated to study the effects of the high expression and knockout of Pls on the gene expression and metabolite synthesis of S. albulus. The high expression of Pls resulted in 598 significantly differentially expressed genes (DEGs) and 425 known differential metabolites, whereas the inactivation of Pls resulted in 868 significant DEGs and 374 known differential metabolites. The expressions of 8 and 35 genes were negatively and positively associated with the Pls expression, respectively. Subsequently, the influence mechanism of the high expression and inactivation of Pls on the ε-PL biosynthetic pathway was elucidated. Twelve metabolites with 30% decreased yield in the high-expression strain of Pls but 30% increased production in the Pls knockout strain were identified. These results demonstrate the influence of Pls on the metabolism of S. albulus. The present work can provide the theoretical basis for improving the production capacity of ε-PL by means of metabolic engineering or developing bioactive substances derived from S. albulus.

AdpA, a developmental regulator, promotes ε-poly-L-lysine biosynthesis in Streptomyces albulus.

BACKGROUND: AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces. RESULTS: In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus. CONCLUSIONS: Here, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

Failure of RQC machinery causes protein aggregation and proteotoxic stress.

Translation of messenger RNAs lacking a stop codon results in the addition of a carboxy-terminal poly-lysine tract to the nascent polypeptide, causing ribosome stalling. Non-stop proteins and other stalled nascent chains are recognized by the ribosome quality control (RQC) machinery and targeted for proteasomal degradation. Failure of this process leads to neurodegeneration by unknown mechanisms. Here we show that deletion of the E3 ubiquitin ligase Ltn1p in yeast, a key RQC component, causes stalled proteins to form detergent-resistant aggregates and inclusions. Aggregation is dependent on a C-terminal alanine/threonine tail that is added to stalled polypeptides by the RQC component, Rqc2p. Formation of inclusions additionally requires the poly-lysine tract present in non-stop proteins. The aggregates sequester multiple cytosolic chaperones and thereby interfere with general protein quality control pathways. These findings can explain the proteotoxicity of ribosome-stalled polypeptides and demonstrate the essential role of the RQC in maintaining proteostasis.

Biotechnological production and application of epsilon-poly-L-lysine (ε-PL): biosynthesis and its metabolic regulation.

Epsilon-poly-L-lysine (ε-PL) is an unusual biopolymer composed of L-lysine produced by several microorganisms, especially by the genus Streptomyces. Due to its excellent antimicrobial activity, good water solubility, high safety, and biodegradable nature, ε-PL with a GRAS status has been widely used in food and pharmaceutical industries. In the past years, studies have focused on the biotechnological production of ɛ-PL, the biosynthetic mechanism of microbial ɛ-PL, and its application. To provide new perspectives from recent advances, the review introduced the methods for the isolation of ɛ-PL producing strains and the biosynthetic mechanism of microbial ɛ-PL. We summarized the strategies for the improvement of ɛ-PL producing strains, including physical and chemical mutagenesis, ribosome engineering and gene engineering, and compared the different metabolic regulation strategies for improving ɛ-PL production, including medium optimization, nutrient supply, pH control, and dissolved oxygen control. Then, the downstream purification methods of ɛ-PL and its recent applications in food and medicine industries were introduced. Finally, we also proposed the potential challenges and the perspectives for the production of ε-PL.

Distribution of ε-Poly-l-Lysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin.

ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems.IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces, also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria.

Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

Poly(lysine) Dendrimers Form Complexes with siRNA and Provide Its Efficient Uptake by Myeloid Cells: Model Studies for Therapeutic Nucleic Acid Delivery.

The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra- and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy.

Not4-dependent translational repression is important for cellular protein homeostasis in yeast.

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.

Translation of poly(A) tails leads to precise mRNA cleavage.

Translation of poly(A) tails leads to mRNA cleavage but the mechanism and global pervasiveness of this "nonstop/no-go" decay process is not understood. Here we performed ribosome profiling (in a yeast strain lacking exosome function) of short 15-18 nucleotides mRNA footprints to identify ribosomes stalled at 3' ends of mRNA decay intermediates. In this background, we found mRNA cleavage extending hundreds of nucleotides upstream of ribosome stalling in poly(A) and predominantly in one reading frame. These observations suggest that decay-triggering endonucleolytic cleavage is closely associated with the ribosome. Surprisingly, ribosomes appeared to accumulate (i.e., stall) in the transcriptome when as few as three consecutive ORF-internal lysine codons were positioned in the A, P, and E sites though significant mRNA degradation was not observed. Endonucleolytic cleavage was found, however, at sites of premature polyadenylation (encoding polylysine) and rescue of the ribosomes stalled at these sites was dependent on Dom34. These results suggest this process may be critical when changes in the polyadenylation site occur during development, tumorigenesis, or when translation termination/recycling is impaired.

Bioresponsive peptide-polysaccharide nanogels - A versatile delivery system to augment the utility of bioactive cargo.

We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or 'nanogel', prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.

Biodegradable Polymers for Gene Delivery.

The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.

ATP-Mediated Transient Behavior of Stomatocyte Nanosystems.

In nature, dynamic processes are ubiquitous and often characterized by adaptive, transient behavior. Herein, we present the development of a transient bowl-shaped nanoreactor system, or stomatocyte, the properties of which are mediated by molecular interactions. In a stepwise fashion, we couple motility to a dynamic process, which is maintained by transient events; namely, binding and unbinding of adenosine triphosphate (ATP). The surface of the nanosystem is decorated with polylysine (PLL), and regulation is achieved by addition of ATP. The dynamic interaction between PLL and ATP leads to an increase in the hydrophobicity of the PLL-ATP complex and subsequently to a collapse of the polymer; this causes a narrowing of the opening of the stomatocytes. The presence of the apyrase, which hydrolyzes ATP, leads to a decrease of the ATP concentration, decomplexation of PLL, and reopening of the stomatocyte. The competition between ATP input and consumption gives rise to a transient state that is controlled by the out-of-equilibrium process.

Partitioning and Enhanced Self-Assembly of Actin in Polypeptide Coacervates.

Biomolecules exist and function in cellular microenvironments that control their spatial organization, local concentration, and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate, and control the local physico-chemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to ∼30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.

Polylysine is a Proteostasis Network-Engaging Structural Determinant.

C-terminal polylysine (PL) can be synthesized from the polyadenine tail of prematurely cleaved mRNAs or when a read-though of a stop codon happens. Due to the highly positive charge, PL stalls in the electrostatically negative ribosomal exit channel. The stalled polypeptide recruits the Ribosome-associated quality control (RQC) complex which processes and extracts the nascent chain. Dysfunction of the RQC leads to the accumulation of PL-tagged proteins, induction of a stress response, and cellular toxicity. Not much is known about the PL-specific aspect of protein quality control. Using quantitative mass spectrometry, we uncovered the post-ribosomal PL-processing machinery in human cytosol. It encompasses key cytosolic complexes of the proteostasis network, such as chaperonin TCP-1 ring complexes (TRiC) and half-capped 19S-20S proteasomes. Furthermore, we found that the nuclear transport machinery associates with PL, which suggests a novel mechanism by which faulty proteins can be compartmentalized in the cell. The enhanced nuclear import of a PL-tagged polypeptide confirmed this implication, which leads to questions regarding the biological rationale behind it.