Aggregation of aberrant proteins is a common pathological hallmark in neurodegeneration such as polyglutamine (polyQ) and other repeat-expansion diseases. Here through overexpression of ataxin3 C-terminal polyQ expansion in Drosophila gut enterocytes, we generated an intestinal obstruction model of spinocerebellar ataxia type3 (SCA3) and reported a new role of nuclear-associated endosomes (NAEs)-the delivery of polyQ to the nucleoplasm. In this model, accompanied by the prominently increased RAB5-positive NAEs are abundant nucleoplasmic reticulum enriched with polyQ, abnormal nuclear envelope invagination, significantly reduced endoplasmic reticulum, indicating dysfunctional nucleocytoplasmic trafficking and impaired endomembrane organization. Consistently, Rab5 but not Rab7 RNAi further decreased polyQ-related NAEs, inhibited endomembrane disorganization, and alleviated disease model. Interestingly, autophagic proteins were enriched in polyQ-related NAEs and played non-canonical autophagic roles as genetic manipulation of autophagic molecules exhibited differential impacts on NAEs and SCA3 toxicity. Namely, the down-regulation of Atg1 or Atg12 mitigated while Atg5 RNAi aggravated the disease phenotypes both in Drosophila intestines and compound eyes. Our findings, therefore, provide new mechanistic insights and underscore the fundamental roles of endosome-centered nucleocytoplasmic trafficking and homeostatic endomembrane allocation in the pathogenesis of polyQ diseases.

Phase separation has emerged as a fundamental principle for organizing viral and cellular membraneless organelles. Although these subcellular compartments have been recognized for decades, their biogenesis and mechanisms of regulation are poorly understood. Here, we investigate the formation of membraneless inclusion bodies (IBs) induced during the infection of a plant rhabdovirus, tomato yellow mottle-associated virus (TYMaV). We generated recombinant TYMaV encoding a fluorescently labeled IB constituent protein and employed live-cell imaging to characterize the intracellular dynamics and maturation of viral IBs in infected Nicotiana benthamiana cells. We show that TYMaV IBs are phase-separated biomolecular condensates and that viral nucleoprotein and phosphoprotein are minimally required for IB formation in vivo and in vitro. TYMaV IBs move along the microfilaments, likely through the anchoring of viral phosphoprotein to myosin XIs. Furthermore, pharmacological disruption of microfilaments or inhibition of myosin XI functions suppresses IB motility, resulting in arrested IB growth and inefficient virus replication. Our study establishes phase separation as a process driving the formation of liquid viral factories and emphasizes the role of the cytoskeletal system in regulating the dynamics of condensate maturation.

Protein misfolding, aggregation, and accumulation cause neurodegenerative disorders. One such disorder, Huntington\'s disease, is caused by an increased number of glutamine-encoding trinucleotide repeats CAG in the first exon of the huntingtin (HTT) gene. Mutant proteins of Htt exon 1 with polyglutamine expansion are prone to aggregation and form pathological inclusion bodies in neurons. Extensive studies have shown that misfolded proteins are cleared by the ubiquitin-proteasome system or autophagy to alleviate their cytotoxicity. Misfolded proteins can form small soluble aggregates or large insoluble inclusion bodies. Previous works have elucidated the role of autophagy in the clearance of misfolded protein aggregates, but autophagic clearance of inclusion bodies remains poorly characterized. Here we use mutant Htt exon 1 with 103 polyglutamine (Htt103QP) as a model substrate to study the autophagic clearance of inclusion bodies in budding yeast. We found that the core autophagy-related proteins were required for Htt103QP inclusion body autophagy. Moreover, our evidence indicates that the autophagy of Htt103QP inclusion bodies is selective. Interestingly, Cue5/Tollip, a known autophagy receptor for aggrephagy, is dispensable for this inclusion body autophagy. From the known selective autophagy receptors in budding yeast, we identified three that are essential for inclusion body autophagy. Amyloid beta peptide (Aβ42) is a major component of amyloid plaques found in Alzheimer\'s disease brains. Interestingly, a similar selective autophagy pathway contributes to the clearance of Aβ42 inclusion bodies in budding yeast. Therefore, our results reveal a novel autophagic pathway specific for inclusion bodies associated with neurodegenerative diseases, which we have termed IBophagy.

Methods for efficient insoluble protein production require further exploration. PagP, an Escherichia coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of recombinant peptides. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. Herein, aggregation \"hot spots\" (HSs) in PagP were analyzed using the web-based software AGGRESCAN, leading to identification of a C-terminal region harboring numerous HSs. Moreover, a proline-rich region was found in the β-strands. Substitution of these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates. Consequently, the absolute yields of recombinant antimicrobial peptides Magainin II, Metchnikowin, and Andropin were increased significantly when expressed in fusion with this refined version of PagP. We describe separation of recombinant target proteins expressed in inclusion bodies fused with the tag. An artificial NHT linker peptide with three motifs was implemented for separation and purification of authentic recombinant antimicrobial peptides. IMPORTANCE Fusion tag-induced formation of inclusion bodies provides a powerful means to express unstructured or toxic proteins. For a given fusion tag, how to enhance the formation of inclusion bodies remains to be explored. Our study illustrated that the aggregation HSs in a fusion tag played important roles in mediating its insoluble expression. Efficient production of inclusion bodies could also be implemented by refining its primary structure to form a more stable β-sheet with higher hydrophobicity. This study provides a promising method for improvement of the insoluble expression of recombinant proteins.

MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins.

Recombinant virus-like particles (VLP) with single capsid protein have been successfully produced through prokaryotic system, but for those with multiple capsid proteins such as the foot-and-mouth disease virus (FMDV), this approach is more challenging. In this study, in vitro assembly of FMDV VLP was investigated with its capsids VP1, VP2 and VP3 separately expressed as inclusion bodies. After extraction and solubilization, three capsids were purified in denatured state through a flow-through model, increasing its purity to 90%. VLP assembly for FMDV was observed after diluting the mixture of denatured capsids in the ration of 1: 1: 1, while no VLP appeared if the separately diluted and refolded capsids were co-incubated. This result suggests certain synergetic interactions exist among the three capsids, which are crucial for FMDV VLP assembly. Sodium chloride and capsid protein concentration both greatly affect the assembling efficiency. After purification through size exclusion chromatography, VLP with similar diameter and morphology as inactivated FMDV were obtained, which elicited high IgG titers and B cell activation when vaccinated in mouse. It could also induce specific humoral and cellular immune responses in splenocytes proliferative experiments. Our study demonstrated the feasibility of in vitro assembling FMDV VLP from inclusion bodies of VP1, VP2 and VP3 for the first time.

Autophagy is emerging as a critical player in host defense against diverse infections, in addition to its conserved function to maintain cellular homeostasis. Strikingly, some pathogens have evolved strategies to evade, subvert or exploit different steps of the autophagy pathway for their lifecycles. Here, we present a new viral mechanism of manipulating autophagy for its own benefit with severe fever with thrombocytopenia syndrome bunyavirus (SFTSV, an emerging high-pathogenic virus) as a model. SFTSV infection triggers autophagy, leading to complete autophagic flux. Mechanistically, we show that the nonstructural protein of SFTSV (NSs) interacts with mTOR, the pivotal regulator of autophagy, by targeting its kinase domain and captures mTOR into viral inclusion bodies (IBs) induced by NSs itself. Furthermore, NSsimpairs mTOR-mediated phosphorylation of unc-51-like kinase 1 (ULK1) at Ser757, disrupting the inhibitory effect of mTOR on ULK1 activity and thus contributing to autophagy induction. Pharmacologic treatment and Beclin-1 knockout experimental results establish that, in turn, autophagy enhances SFTSV infection and propagation. Moreover, the minigenome reporter system reveals that SFTSV ribonucleoprotein (the transcription and replication machinery) activity can be bolstered by autophagy. Additionally, we found that the NSs proteins of SFTSV-related bunyaviruses have a conserved function of targeting mTOR. Taken together, we unravel a viral strategy of inducing pro-viral autophagy by interacting with mTOR, sequestering mTOR into IBs and hence provoking the downstream ULK1 pathway, which presents a new paradigm for viral manipulation of autophagy and may help inform future development of specific antiviral therapies against SFTSV and related pathogens.

Antigen-binding variable domains of the H chain of heavy-chain antibodies (VHHs), also known as nanobodies (Nbs), are of great interest in imaging technique, disease prevention, diagnosis, and therapy. High-level expression of soluble Nbs is very important for its industrial production. In this study, we optimized the expression system of anti-green fluorescent protein (GFP) VHHs with three different signal peptides (SPs), outer-membrane protein A (OmpA), pectate lyase B (PelB), and L-asparaginase II SP (L-AsPsII), in different Escherichia coli strains via isopropyl β-D-thiogalactoside (IPTG) induction and auto-induction, respectively. The solubility of recombinant anti-GFP VHHs with PelB or OmpA was significantly enhanced to the same extent by IPTG induction and auto-induction in BL21 (DE3) E. coli strain and the maximum yield of target protein reached approximately 0.4 mg/l in a shake flask. The binding activity of recombinant anti-GFP VHHs was also confirmed to be retained by native-polyacrylamide gel electrophoresis (PAGE). These results suggest that SPs like OmpA and PelB could efficiently improve the recombinant anti-GFP VHH solubility without changing its bioactivity, providing a novel strategy to optimize the E. coli expression system of soluble VHHs, and lay the foundation for the industrial production of soluble recombinant anti-GFP VHHs and the research of other VHHs in the future.

5-Hydroxymethylfurfural oxidase (HMFO) can catalyze both hydroxyl and aldehyde oxidations. It catalyzes 5-hydroxymethylfurfural into 2,5-furandicarboxylic acid. However, the application of HMFO encountered two problems: the expressed HMFO in Escherichia coli. is largely in the form of inclusion bodies, and the by-product of H2O2 has a negative effect on HMFO stability. To solve these problems, recombinant HMFO was generated by fusing the C-terminus to an elastin-like polypeptide (ELP). ELP-HMFO can be expressed with significantly reduced inclusion bodies. ELP-HMFO exhibited improved stability and tolerance toward H2O2. Further recombination is carried out by fusing the N-terminus of HMFO to a glutamic acid-rich leucine zipper motif (ZE). Similarly, recombinant catalase (CAT) is generated by fusing the N-terminus to ELP and fusing the C-terminus to an arginine-rich leucine zipper motif (ZR). ELP-HMFO-ZE can interact specifically with ZR-CAT-ELP, ascribing to the coiled-coil association of ZE and ZR. ELP-HMFO-ZE#ZR-CAT-ELP coordinates the respective catalytic activities of the two enzymes. ELP-HMFO-ZE catalyzes the oxidation of HMF, and the generated hydrogen peroxide is decomposed by ZR-CAT-ELP into H2O and oxygen. During the oxidation of HMF, the cofactor FAD of HMFO is reduced, and molecular oxygen is needed to reoxidize the reduced FAD. The evolved oxygen from the decomposing of H2O2 can just meet the requirement, which can be diffused efficiently from ZR-CAT-ELP to ELP-HMFO-ZE due to the short distance between the two enzymes.

The current standard for the diagnosis of COVID-19 is the nucleic acid test of SARS-CoV-2 RNA, however, virus antibody detection has the advantages of convenient sample collection, high throughout, and low cost. When combining detection with nucleic acid detection, antibody detection can effectively compensate for nucleic acid detection. Virus infection always induce high antibody titer against SARS-CoV-2 nucleocapsid protein (N protein), which can be used to detect COVID-19 at both infected and convalescent patients. In this study we reported the expression and purification of N protein in E.coli from inclusion bodies by a combination of two cation exchange chromatography, and the yield of N protein was around 50 mg/L fermentation broth with more than 90% purity. A corresponding colloidal gold detection kit prepared with our purified N protein was used to verify the efficiency and accuracy our N protein in antibody detection method. Of the 58 COVID-19 PCR positive patients\' inactivated serum samples, 40 samples were IgM positive (69.0%), and 42 samples were IgG positive (72.4%), and all 95 COVID-19 negative patients\' inactivated serum samples were both IgM and IgG negative. Our results indicates that the refolded soluble N protein could be used for the preliminary detection of IgG and IgM antibodies against SARS-CoV- 2.