OTUB1 regulates ferroptosis to inhibit myoblast differentiation into myotubes by deubiquitinating P62.

As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death (PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.

N6-methyladenosine-modified SENP1, identified by IGF2BP3, is a novel molecular marker in acute myeloid leukemia and aggravates progression by activating AKT signal via de-SUMOylating HDAC2.

BACKGROUND: Elevated evidence suggests that the SENPs family plays an important role in tumor progression. However, the role of SENPs in AML remains unclear. METHODS: We evaluated the expression pattern of SENP1 based on RNA sequencing data obtained from OHSU, TCGA, TARGET, and MILE datasets. Clinical samples were used to verify the expression of SENP1 in the AML cells. Lentiviral vectors shRNA and sgRNA were used to intervene in SENP1 expression in AML cells, and the effects of SENP1 on AML proliferation and anti-apoptosis were detected using in vitro and in vivo models. Chip-qPCR, MERIP-qPCR, CO-IP, RNA pulldown, and dual-luciferase reporter gene assays were used to explore the regulatory mechanisms of SNEP1 in AML. RESULTS: SENP1 was significantly upregulated in high-risk AML patients and closely related to poor prognosis. The AKT/mTOR signaling pathway is a key downstream pathway that mediates SENP1's regulation of AML proliferation and anti-apoptosis. Mechanistically, the CO-IP assay revealed binding between SENP1 and HDAC2. SUMO and Chip-qPCR assays suggested that SENP1 can desumoylate HDAC2, which enhances EGFR transcription and activates the AKT pathway. In addition, we found that IGF2BP3 expression was upregulated in high-risk AML patients and was positively correlated with SENP1 expression. MERIP-qPCR and RIP-qPCR showed that IGF2BP3 binds SENP1 3-UTR in an m6A manner, enhances SENP1 expression, and promotes AKT pathway conduction. CONCLUSIONS: Our findings reveal a distinct mechanism of SENP1-mediated HDAC2-AKT activation and establish the critical role of the IGF2BP3/SENP1signaling axis in AML development.

Norepinephrine Drives Sleep Fragmentation Activation of Asparagine Endopeptidase, Locus Ceruleus Degeneration, and Hippocampal Amyloid-ß42 Accumulation.

Chronic sleep disruption (CSD), from insufficient or fragmented sleep and is an important risk factor for Alzheimer's disease (AD). Underlying mechanisms are not understood. CSD in mice results in degeneration of locus ceruleus neurons (LCn) and CA1 hippocampal neurons and increases hippocampal amyloid-ß42 (Aß42), entorhinal cortex (EC) tau phosphorylation (p-tau), and glial reactivity. LCn injury is increasingly implicated in AD pathogenesis. CSD increases NE turnover in LCn, and LCn norepinephrine (NE) metabolism activates asparagine endopeptidase (AEP), an enzyme known to cleave amyloid precursor protein (APP) and tau into neurotoxic fragments. We hypothesized that CSD would activate LCn AEP in an NE-dependent manner to induce LCn and hippocampal injury. Here, we studied LCn, hippocampal, and EC responses to CSD in mice deficient in NE [dopamine ß-hydroxylase (Dbh)-/-] and control male and female mice, using a model of chronic fragmentation of sleep (CFS). Sleep was equally fragmented in Dbh -/- and control male and female mice, yet only Dbh -/- mice conferred resistance to CFS loss of LCn, LCn p-tau, and LCn AEP upregulation and activation as evidenced by an increase in AEP-cleaved APP and tau fragments. Absence of NE also prevented a CFS increase in hippocampal AEP-APP and Aß42 but did not prevent CFS-increased AEP-tau and p-tau in the EC. Collectively, this work demonstrates AEP activation by CFS, establishes key roles for NE in both CFS degeneration of LCn neurons and CFS promotion of forebrain Aß accumulation, and, thereby, identifies a key molecular link between CSD and specific AD neural injuries.

Targeting Ser78 phosphorylation of Hsp27 achieves potent antiviral effects against enterovirus A71 infection.

A positive-sense (+) single-stranded RNA (ssRNA) virus (e.g. enterovirus A71, EV-A71) depends on viral polypeptide translation for initiation of virus replication after entry. We reported that EV-A71 hijacks Hsp27 to induce hnRNP A1 cytosol redistribution to initiate viral protein translation, but the underlying mechanism is still elusive. Here, we show that phosphorylation-deficient Hsp27-3A (Hsp27S15/78/82A) and Hsp27S78A fail to translocate into the nucleus and induce hnRNP A1 cytosol redistribution, while Hsp27S15A and Hsp27S82A display similar effects to the wild type Hsp27. Furthermore, we demonstrate that the viral 2A protease (2Apro) activity is a key factor in regulating Hsp27/hnRNP A1 relocalization. Hsp27S78A dramatically decreases the IRES activity and viral replication, which are partially reduced by Hsp27S82A. However, Hsp27S15A displays the same activity as the wild-type Hsp27. Peptide S78 potently suppresses EV-A71 protein translation and reproduction through blockage of EV-A71-induced Hsp27 phosphorylation and Hsp27/hnRNP A1 relocalization. A point mutation (S78A) on S78 impairs its inhibitory functions on Hsp27/hnRNP A1 relocalization and viral replication. Taken together, we demonstrate the importance of Ser78 phosphorylation of Hsp27 regulated by virus infection in nuclear translocation, hnRNP A1 cytosol relocation, and viral replication, suggesting a new path (such as peptide S78) for target-based antiviral strategy.

Adaptive substitutions at two amino acids of HCPro modify its functional properties to separately increase the virulence of a potyviral chimera.

We had previously reported that a plum pox virus (PPV)-based chimera that had its P1-HCPro bi-cistron replaced by a modified one from potato virus Y (PVY) increased its virulence in some Nicotiana benthamiana plants, after mechanical passages. This correlated with the natural acquisition of amino acid substitutions in several proteins, including in HCPro at either position 352 (Ile→Thr) or 454 (Leu→Arg), or of mutations in non-coding regions. Thr in position 352 is not found among natural potyviruses, while Arg in 454 is a reversion to the native PVY HCPro amino acid. We show here that both mutations separately contributed to the increased virulence observed in the passaged chimeras that acquired them, and that Thr in position 352 is no intragenic suppressor to a Leu in position 454, because their combined effects were cumulative. We demonstrate that Arg in position 454 improved HCPro autocatalytic cleavage, while Thr in position 352 increased its accumulation and the silencing suppression of a reporter in agropatch assays. We assessed infection by four cloned chimera variants expressing HCPro with none of the two substitutions, one of them or both, in wild-type versus DCL2/4-silenced transgenic plants. We found that during infection, the transgenic context of altered small RNAs affected the accumulation of the four HCPro variants differently and hence, also infection virulence.

OTUB1 contributes to the stability and function of Influenza A virus NS2.

The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.

A new point mutation in the HC-Pro of potato virus Y is involved in tobacco vein necrosis.

Tobacco vein necrosis (TVN) is a complex phenomenon regulated by different genetic determinants mapped in the HC-Pro protein (amino acids N330, K391 and E410) and in two regions of potato virus Y (PVY) genome, corresponding to the cytoplasmic inclusion (CI) protein and the nuclear inclusion protein a-protease (NIa-Pro), respectively. A new determinant of TVN was discovered in the MK isolate of PVY which, although carried the HC-Pro determinants associated to TVN, did not induce TVN. The HC-Pro open reading frame (ORF) of the necrotic infectious clone PVY N605 was replaced with that of the non-necrotic MK isolate, which differed only by one amino acid at position 392 (T392 instead of I392). The cDNA clone N605_MKHCPro inoculated in tobacco induced only weak mosaics at the systemic level, demostrating that the amino acid at position 392 is a new determinant for TVN. No significant difference in accumulation in tobacco was observed between N605 and N605_MKHCPro. Since phylogenetic analyses showed that the loss of necrosis in tobacco has occurred several times independently during PVY evolution, these repeated evolutions strongly suggest that tobacco necrosis is a costly trait in PVY.

SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5.

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

SENP1-Mediated deSUMOylation Regulates the Tumor Remodeling of Glioma Stem Cells Under Hypoxic Stress.

Objectives: This study aimed to investigate the effect of specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1)-mediated deSUMOylation on the malignant behavior of glioma stem cells (GSCs) under hypoxia conditions and evaluate the clinical value of prevention in glioma patients. Introductions: Under hypoxic conditions, upregulated hypoxia-inducible factor 1α (HIF1α) expression in GSCs activates Wnt/ß-catenin signaling pathways, which provide rich nutritional support for glioblastoma (GBM). SENP1-mediated deSUMOylation stabilizes the expression of HIF1α and ß-catenin, leading to the occurrence of GSCs-initiated tumorigenesis. Targeting SENP1-mediated deSUMOylation may suppress the malignancy of GSCs and disrupt GBM progression. Methods: The expression of SENP1 in different World Health Organization grades was observed by immunohistochemistry and western blot. Lentivirus-packaged SENP1shRNA downregulated the expression of SENP1 in GSCs, and the downregulated results were verified by western blotting and polymerase chain reaction. The effects of LV-SENP1shRNA on the migration and proliferation of GSCs were detected by scratch and cloning experiments. The effect of LV-SENP1shRNA on the tumor formation ability of GSCs was observed in nude mice. Immunoprecipitation clarified the mechanism of SENP1 regulating the malignant behavior of GSCs under hypoxia. The correlation between the expression level of SENP1 and the survival of glioma patients was determined by statistical analysis. Results: SENP1 expression in GSCs derived from clinical samples was upregulated in GBM. SUMOylation was observed in GSCs in vitro, and deSUMOylation, accompanied by an increase in SENP1 expression, was induced by hypoxia. SENP1 expression was downregulated in GSCs with lentivirus-mediated stable transfection, which attenuated the proliferation and differentiation of GSCs, thus diminishing tumorigenesis. Mechanistically, HIF1α induced activation of Wnt/ß-catenin, which depended on SENP1-mediated deSUMOylation, promoting GSC-driven GBM growth under the hypoxia microenvironment. Conclusion: Our findings indicate that SENP1-mediated deSUMOylation as a feature of GSCs is essential for GBM maintenance, suggesting that targeting SENP1 against GSCs may effectively improve GBM therapeutic efficacy.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Helper Component-Proteinase of Triticum Mosaic Virus Is a Viral Determinant of Wheat Curl Mite Transmission.

Triticum mosaic virus (TriMV; genus Poacevirus; family Potyviridae) is an economically important virus in the Great Plains region of the United States. TriMV is transmitted by the wheat curl mite (Aceria tosichella) Type 2 genotype but not by Type 1. Helper component-proteinase (HC-Pro) is a vector transmission determinant for several potyvirids, but the role of HC-Pro in TriMV transmission is unknown. In this study, we examined the requirement of the HC-Pro cistron of TriMV for wheat curl mite (Type 2) transmission through deletion and point mutations and constructing TriMV chimeras with heterologous HC-Pros from other potyvirids. TriMV with complete deletion of HC-Pro failed to be transmitted by wheat curl mites at detectable levels. Furthermore, TriMV chimeras with heterologous HC-Pros from aphid-transmitted turnip mosaic virus and tobacco etch virus, or wheat curl mite-transmitted wheat streak mosaic virus, failed to be transmitted by wheat curl mites. These data suggest that heterologous HC-Pros did not complement TriMV for wheat curl mite transmission. A decreasing series of progressive nested in-frame deletions at the N-terminal region of HC-Pro comprising amino acids 3 to 125, 3 to 50, 3 to 25, 3 to 15, 3 to 8, and 3 and 4 abolished TriMV transmission by wheat curl mites. Additionally, mutation of conserved His20, Cys49, or Cys52 to Ala in HC-Pro abolished TriMV transmissibility by wheat curl mites. These data suggest that the N-terminal region of HC-Pro is crucial for TriMV transmission by wheat curl mites. Collectively, these data demonstrate that the HC-Pro cistron of TriMV is a viral determinant for wheat curl mite transmission.

The Transpeptidase Sortase A Binds Nucleic Acids and Mediates Mammalian Cell Labeling.

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRß.

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction. Vascular smooth muscle cells (VSMCs), the main components of atherosclerotic plaque, switch from contractile to synthetic phenotypes during atherogenesis. Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis, and it can be reversely regulated by deubiquitinases. However, the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated. In this study, RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases, which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch. Further in vivo studies using Apoe-/- mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype. Moreover, VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro. Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation, adhesion, and proliferation. Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated. Mechanistically, we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRß with its catalytic triad, thereby reducing the K48-linked ubiquitylation of PDGFRß. Inhibiting OTUB1 in VSMCs could promote PDGFRß degradation via the ubiquitin-proteasome pathway, so it was beneficial in preventing VSMCs' phenotype switch. These findings revealed that knocking down OTUB1 ameliorated VSMCs' phenotype switch and atherosclerosis progression, indicating that OTUB1 could be a valuable translational therapeutic target in the future.

Senp7 deficiency impairs lipid droplets maturation in white adipose tissues via Plin4 deSUMOylation.

Lipid metabolism is important for the maintenance of physiological homeostasis. Several members of the small ubiquitin-like modifier (SUMO)-specific protease (SENP) family have been reported as the regulators of lipid homeostasis. However, the function of Senp7 in lipid metabolism remains unclear. In this study, we generated both conventional and adipocyte-specific Senp7 KO mice to characterize the role of Senp7 in lipid metabolism homeostasis. Both Senp7-deficient mice displayed reduced white adipose tissue mass and decreased size of adipocytes. By analyzing the lipid droplet morphology, we demonstrated that the lipid droplet size was significantly smaller in Senp7-deficient adipocytes. Mechanistically, Senp7 could deSUMOylate the perilipin family protein Plin4 to promote the lipid droplet localization of Plin4. Our results reveal an important role of Senp7 in the maturation of lipid droplets via Plin4 deSUMOylation.

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase.

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2.

SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.

Hotspot site microenvironment in the deubiquitinase OTUB1 drives its stability and aggregation.

Lewy bodies (LB) are aberrant protein accumulations observed in the brain cells of individuals affected by Parkinson's disease (PD). A comprehensive analysis of LB proteome identified over a hundred proteins, many co-enriched with α-synuclein, a major constituent of LB. Within this context, OTUB1, a deubiquitinase detected in LB, exhibits amyloidogenic properties, yet the mechanisms underlying its aggregation remain elusive. In this study, we identify two critical sites in OTUB1-namely, positions 133 and 173-that significantly impact its amyloid aggregation. Substituting alanine at position 133 and lysine at position 173 enhances both thermodynamic and kinetic stability, effectively preventing amyloid aggregation. Remarkably, lysine at position 173 demonstrates the highest stability without compromising enzymatic activity. The increased stability and inhibition of amyloid aggregation are attributed mainly to the changes in the specific microenvironment at the hotspot. In our exploration of the in-vivo co-occurrence of α-synuclein and OTUB1 in LB, we observed a synergistic modulation of each other's aggregation. Collectively, our study unveils the molecular determinants influencing OTUB1 aggregation, shedding light on the role of specific residues in modulating aggregation kinetics and structural transition. These findings contribute valuable insights into the complex interplay of amino acid properties and protein aggregation, with potential implications for understanding broader aspects of protein folding and aggregation phenomena.

Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection.

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K124 and K182) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K50) in KITC motif, K124, and K182 were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HCELR. The mutant PVY-HCELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HCELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HCELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HCELR mutant was a promising mild mutant for cross-protection to control PVY.

A flexible 'plug and play' bicistronic construct and its application in the screening of protein expression system in Escherichia coli.

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.