Journal: Viruses

Article Title: HBV Infection Drives PSMB5-Dependent Proteasomal Activation in Humanized Mice and HBV-Associated HCC

doi: 10.3390/v17111454

Figure Lengend Snippet: HBV infection enhances PSMB5 expression in liver-humanized mice. ( A ) 10-year survival analysis of low (bottom 75% of TPM) and high (top 25% of TPM) PSMA1 expressing HCC patients using GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses . HBV infection increases PSMB5 gene expression levels of liver humanized mice livers. ( B ) Schematic overview of HBV infection in uPA-NOG liver-humanized mice. ( C ) Human albumin levels in mouse serum, 8 weeks post-transplantation ( D ) H&E staining of mouse liver tissue. ( E ) Anti-human mitochondria staining indicating engrafted human hepatocytes after 12 weeks of infection. ( F ) Relative mRNA expression of 26S proteasome subunits in livers of HBV-infected vs. non-infected mice ( n = 4/group). TPM: Transcripts per million. Scale bar: 100 μm.

Article Snippet: The plasmid targeting the human PSMB5 gene (#117073) and the LentiCRISPRv2 plasmid with blasticidin selection (#83480) were obtained from Addgene (Watertown, MA, USA).

Techniques: Infection, Expressing, Gene Expression, Transplantation Assay, Staining

Journal: Viruses

Article Title: HBV Infection Drives PSMB5-Dependent Proteasomal Activation in Humanized Mice and HBV-Associated HCC

doi: 10.3390/v17111454

Figure Lengend Snippet: Functional consequences of PSMB5 knockout in human THP-1 cells. ( A ) Ten-year survival analysis of low (bottom 75% of TPM) and high (top 25% of TPM) PSMB5 expressing HCC patients using GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses . ( B ) PSMB5KO cells accumulate ubiquitinated proteins due to decreased protein degradation. Western blot analysis showing accumulation of ubiquitinated proteins in PSMB5-deficient (KO) THP-1 cells compared with wild-type (WT) controls on day 7. ( C ) Flow cytometric analysis of MHC I surface expression in WT and PSMB5KO THP-1cells at days 7 and day 14 post-transduction. ( D ) Relative mRNA expression levels of antigen-processing pathway genes in WT ( n = 3) and PSMB5-KO ( n = 3) THP-1 cells. WT: wild-type control; KO: PSMB5 knockout. * p < 0.05.

Article Snippet: The plasmid targeting the human PSMB5 gene (#117073) and the LentiCRISPRv2 plasmid with blasticidin selection (#83480) were obtained from Addgene (Watertown, MA, USA).

Techniques: Functional Assay, Knock-Out, Expressing, Gene Expression, Western Blot, Transduction, Control

Journal: Viruses

Article Title: HBV Infection Drives PSMB5-Dependent Proteasomal Activation in Humanized Mice and HBV-Associated HCC

doi: 10.3390/v17111454

Figure Lengend Snippet: Elevated β5 subunit expression and proteasome activity in HBV-infected human tissues. ( A ) Differential expression of PSMB5 gene in tumor and corresponding normal tissue from patients with HCC using GEPIA . HBV infected patients have higher levels of β5 protein expression and proteasome activity levels. Liver and serum samples of HBV, HCC and HBV-induced HCC patients were collected; ( B ) proteasome subunit β5 protein expression and ( C ) proteasome activity were analyzed with ELISA methods. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The plasmid targeting the human PSMB5 gene (#117073) and the LentiCRISPRv2 plasmid with blasticidin selection (#83480) were obtained from Addgene (Watertown, MA, USA).

Techniques: Expressing, Activity Assay, Infection, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: Establishing the covalently reactive peptide/protein pair SnoopTag and SnoopCatcher. (A) Spontaneous isopeptide bond formation between Lys and Asn, releasing ammonia. (B) Cartoon of splitting RrgA D4 domain [based on Protein Data Bank (PDB) ID code 2WW8] to make SnoopTag and SnoopCatcher. Reactive residues are in cyan. (C) SnoopTag-MBP reaction with SnoopCatcher, each at 10 µM, after 2 h at 25 °C analyzed by SDS/PAGE with Coomassie staining, alongside controls with Ala mutation of SnoopTag’s reactive Lys (KA) or SnoopCatcher’s reactive Asn (NA). (D) Isopeptide bond formation between SnoopTag peptide and SnoopCatcher shown by mass spectrometry. (E) Time course of SnoopTag reaction with a 1:1 or 2:1 ratio of SnoopCatcher to SnoopTag-MBP, tested as in C. (F) Time course of SnoopCatcher reaction with a 1:1, 2:1, or 4:1 ratio of SnoopTag-MBP to SnoopCatcher, tested as in C. Error bars are mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Mutagenesis, Mass Spectrometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Programmable polyproteams built using twin peptide superglues

doi: 10.1073/pnas.1519214113

Figure Lengend Snippet: SnoopTag and SnoopCatcher reaction conditions. (A) Point mutations to make SnoopCatcher. From the C-terminal domain of RrgA in cartoon format (based on PDB ID code 2WW8), residues mutated are shown in space-fill with carbons in cyan (G842 was changed to T and D848 to G). The Lys, Asn, and Glu involved in isopeptide bond formation are shown in stick format in yellow and the rest of SnoopTag colored magenta. (B) Sample gel to test for quantitative reaction of SnoopTag. Shown are 10 µM SnoopTag-MBP and 20 µM SnoopCatcher alone or mixed for 32 min (triplicate samples) before SDS/PAGE with Coomassie staining. (C) Sample gel to test for quantitative reaction of SnoopCatcher. Shown are 10 µM SnoopCatcher and 40 µM SnoopTag-MBP alone or mixed for the indicated times (triplicate samples) before SDS/PAGE with Coomassie staining. (D) Buffer dependence of SnoopTag/SnoopCatcher reaction. We incubated 10 µM SnoopTag-MBP with 10 µM SnoopCatcher at pH 8.0 for 15 min at 25 °C in the indicated buffer and analyzed the samples by SDS/PAGE with Coomassie staining. (E) TMAO dependence of SnoopTag/SnoopCatcher reaction tested as in D. Error bars are all mean ±1 SD; n = 3. Some error bars are too small to be visible.

Article Snippet: Constructs were initially cloned into chemically competent E. coli DH5α (Life Technologies). pET28a SpyTag-MBP (Addgene plasmid ID 35050), GST-BirA, and pDEST14-SpyCatcher (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JQ478411","term_id":"380294102","term_text":"JQ478411"}} JQ478411 , Addgene plasmid ID 35044) have been described ( 26 ). pET28a SnoopCatcher (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KU500646","term_id":"985768551","term_text":"KU500646"}} KU500646 , Addgene plasmid ID 72322) was generated by DNAWorks primer-mediated assembly from residues 749–860 of S. pneumoniae adhesin RrgA (numbering based on PDB ID code 2WW8) ( 28 ), digested with HindIII and NdeI and subcloned into pET28a.

Techniques: SDS Page, Staining, Incubation