Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.

Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo . We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo .

Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.

Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo. We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo.

Rapid purification of rabbit immunoglobulins using a single-step, negative-selection chromatography.

Custom polyclonal antibodies raised in rabbits are routinely used in immunoblotting and other protein analysis techniques. Custom rabbit polyclonal antisera are generally purified using immunoaffinity or Protein A-affinity chromatography; however, these methods require harsh elution conditions that can compromise the antigen binding efficacy. We evaluated the utility of Melon™ Gel chromatography for purification of IgG from crude rabbit serum. We show that Melon Gel-purified rabbit IgGs are active and perform well in immunoblotting. In summary, the Melon Gel method is a rapid, one-step, negative-selection approach that can be employed in either preparative or small-scale format to purify IgG from crude rabbit serum without the need for denaturing eluent.

Structure and functional determinants of Rad6-Bre1 subunits in the histone H2B ubiquitin-conjugating complex.

The conserved complex of the Rad6 E2 ubiquitin-conjugating enzyme and the Bre1 E3 ubiquitin ligase catalyzes histone H2B monoubiquitination (H2Bub1), which regulates chromatin dynamics during transcription and other nuclear processes. Here, we report a crystal structure of Rad6 and the non-RING domain N-terminal region of Bre1, which shows an asymmetric homodimer of Bre1 contacting a conserved loop on the Rad6 'backside'. This contact is distant from the Rad6 catalytic site and is the location of mutations that impair telomeric silencing in yeast. Mutational analyses validated the importance of this contact for the Rad6-Bre1 interaction, chromatin-binding dynamics, H2Bub1 formation and gene expression. Moreover, the non-RING N-terminal region of Bre1 is sufficient to confer nucleosome binding ability to Rad6 in vitro. Interestingly, Rad6 P43L protein, an interaction interface mutant and equivalent to a cancer mutation in the human homolog, bound Bre1 5-fold more tightly than native Rad6 in vitro, but showed reduced chromatin association of Bre1 and reduced levels of H2Bub1 in vivo. These surprising observations imply conformational transitions of the Rad6-Bre1 complex during its chromatin-associated functional cycle, and reveal the differential effects of specific disease-relevant mutations on the chromatin-bound and unbound states. Overall, our study provides structural insights into Rad6-Bre1 interaction through a novel interface that is important for their biochemical and biological responses.

Quantitative Assessment of Histone H2B Monoubiquitination in Yeast Using Immunoblotting.

Studies in Saccharomyces cerevisiae and Schizosaccharomyces pombe have enhanced our understanding of the regulation and functions of histone H2B monoubiquitination (H2Bub1), a key epigenetic marker with important roles in transcription and other processes. The detection of H2Bub1 in yeasts using immunoblotting has been greatly facilitated by the commercial availability of antibodies against yeast histone H2B and the cross-reactivity of an antibody raised against monoubiquitinated human H2BK120. These antibodies have obviated the need to express epitope-tagged histone H2B to detect H2Bub1 in yeasts. Here, we provide a step-by-step protocol and best practices for the quantification of H2Bub1 in yeast systems, from cell extract preparation to immunoblotting using the commercially available antibodies. We demonstrate that the commercial antibodies can effectively and accurately detect H2Bub1 in S. cerevisiae and S. pombe. Further, we show that the C-terminal epitope-tagging of histone H2B alters the steady-state levels of H2Bub1 in yeast systems. We report a sectioned blot probing approach combined with the serial dilution of protein lysates and the use of reversibly stained proteins as loading controls that together provide a cost-effective and sensitive method for the quantitative evaluation of H2Bub1 in yeast.

Mutations of Rad6 E2 ubiquitin-conjugating enzymes at alanine-126 in helix-3 affect ubiquitination activity and decrease enzyme stability.

Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance, and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair, and protein degradation was reported over 2 decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6 in addition to disorganizing the global structure of the protein to decrease its stability in vivo. We also show that helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. Providing insights into their ubiquitination defects, we determined helix-3 A126 mutations impair the initial ubiquitin charging and the final discharging steps during substrate ubiquitination by Rad6. In summary, our studies reveal that the conserved helix-3 is a crucial structural constituent that controls the organization of catalytic pockets, enzymatic activities, and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.

Unification and extensive diversification of M/Orf3-related ion channel proteins in coronaviruses and other nidoviruses.

The coronavirus, Severe Acute Respiratory Syndrome (SARS)-CoV-2, responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, has emphasized the need for a better understanding of the evolution of virus-host interactions. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) implicated in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. We present computational evidence that these viral families might utilize specific conserved polar residues to constitute an aqueous pore within the membrane-spanning region. We reconstruct an evolutionary history of these families and objectively establish the common origin of the M proteins of CoVs and Toroviruses. We also show that the divergent ORF3 clade (ORF3a/ORF3b/ORF3c/ORF5 families) represents a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a hypothesis for their role in virion assembly of CoVs, ToroVs, and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly and membrane budding. We show that M and ORF3 are under different evolutionary pressures; in contrast to the slow evolution of M as core structural component, the ORF3 clade is under selection for diversification, which suggests it might act at the interface with host molecules and/or immune attack.

Unification of the M/ORF3-related proteins points to a diversified role for ion conductance in pathogenesis of coronaviruses and other nidoviruses.

The new coronavirus, SARS-CoV-2, responsible for the COVID-19 pandemic has emphasized the need for a better understanding of the evolution of virus-host conflicts. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) and involved in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. By sequence analysis and structural modeling, we show that these viral families utilize specific conserved polar residues to constitute an ion-conducting pore in the membrane. We reconstruct the evolutionary history of these families, objectively establish the common origin of the M proteins of CoVs and Toroviruses. We show that the divergent ORF3a/ORF3b/ORF5 families represent a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a model for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly, membrane fusion and budding. We show that the M and ORF3 are under differential evolutionary pressures; in contrast to the slow evolution of M as core structural component, the CoV-ORF3 clade is under selection for diversification, which indicates it is likely at the interface with host molecules and/or immune attack. IMPORTANCE: Coronaviruses (CoVs) have become a major threat to human welfare as the causative agents of several severe infectious diseases, namely Severe Acute Respiratory Syndrome (SARS), Middle Eastern Respiratory Syndrome (MERS), and the recently emerging human coronavirus disease 2019 (COVID-19). The rapid spread, severity of these diseases, as well as the potential re-emergence of other CoV-associated diseases have imposed a strong need for a thorough understanding of function and evolution of these CoVs. By utilizing robust domain-centric computational strategies, we have established homologous relationships between many divergent families of CoV proteins, including SARS-CoV/SARS-CoV-2 ORF3a, MERS-CoV ORF5, proteins from both beta-CoVs (ORF3c) and alpha-CoVs (ORF3b), the typical CoV Matrix proteins, and many distant homologs from other nidoviruses. We present evidence that they are active ion channel proteins, and the Cov-specific ORF3 clade proteins are under selection for rapid diversification, suggesting they might have been involved in interfering host molecules and/or immune attack.

Role of N-terminal region of Escherichia coli maltodextrin glucosidase in folding and function of the protein.

Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-Terminal region of MalZ. The crystal structure of this enzyme has been reported in the present communication. Based on the crystallographic structure, it has been interpreted that the N-terminal region of the enzyme (Met1-Phe131) might be unstructured or flexible. To understand the role of the N-terminal region of MalZ in its enzymatic activity, and overall stability, a truncated version (Ala111-His616) of MalZ was created. The truncated version failed to fold into an active enzyme both in E. coli cytosol and in vitro even with the assistance of chaperonins GroEL and GroES. Furthermore, the refolding effort of N-truncated MalZ in the presence of isolated N-terminal domain didn't succeed. Our studies suggest that while the structural rigidity or orientation of the N-terminal region of the MalZ protein may not be essential for its stability and function, but the said domain is likely to play an important role in the formation of the native structure of the protein when present as an integral part of the protein.