Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway.

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.

Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV.

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2.

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture.

Serum albumin physically interacts with fatty acids, small molecules, metal ions, and several other proteins. Binding with a plethora of bioactive substances makes it a critical transport molecule. Albumin also scavenges the reactive oxygen species that are harmful to cell survival. These properties make albumin an excellent choice to promote cell growth and maintain a variety of eukaryotic cells under in vitro culture environment. Furthermore, purified recombinant human serum albumin is mostly free from impurities and modifications, providing a perfect choice as an additive in cell and tissue culture media while avoiding any regulatory constraints. This review discusses key features of human serum albumin implicated in cell growth and survival under in vitro conditions.

Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.

Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.

Phenyl Dihydrouracil: An Alternative Cereblon Binder for PROTAC Design.

Thalidomide and its analogues are frequently used in PROTAC design. However, they are known to be inherently unstable, undergoing hydrolysis even in commonly utilized cell culture media. We recently reported that phenyl glutarimide (PG)-based PROTACs displayed improved chemical stability and, consequently, improved protein degradation efficacy and cellular potency. Our optimization efforts, aiming to further improve the chemical stability and eliminate the racemization-prone chiral center in PG, led us to the development of phenyl dihydrouracil (PD)-based PROTACs. Here we describe the design and synthesis of LCK-directing PD-PROTACs and compare their physicochemical and pharmacological properties to those of the corresponding IMiD and PG analogues.

From PROTAC to inhibitor: Structure-guided discovery of potent and orally bioavailable BET inhibitors.

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development.

Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite.

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol(-1) was observed between WT-protein and W101A mutant.

Dot-Blotting: A Quick Method for Expression Analysis of Recombinant Proteins.

Expressing recombinant proteins in heterologous host cells is a prerequisite for purification and other downstream processes. Cell cultures require a protein expression test to optimize incubation time, temperature, and additives (like chemical inducers) to identify the best growth conditions with maximum recombinant protein yield. However, running SDS-PAGE followed by western blotting is cumbersome and results are not quick. Here, I describe a simple protocol to quickly check the presence of recombinant protein in cell cultures using a dot-blot experiment. The cells can be rapidly lysed and directly spotted on the nitrocellulose membrane. Then, the membrane is incubated with a horseradish peroxidase (HRP) conjugated antibody raised against the affinity tag present on the recombinant protein to confirm the protein expression by chemiluminescence. It takes less than an hour to get results. This method rapidly investigates recombinant protein expression in different cell lines and tests other variables. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protein expression analysis for eukaryotic systems Basic Protocol 2: Protein expression analysis for bacterial systems.

A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation.

In plants, selfish genetic elements, including retrotransposons and DNA viruses, are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp that are synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV's choice of initiating nucleotide, RDR2's initiation 1-2 nt internal to Pol IV transcript ends and RDR2's terminal transferase activity collectively yield a code that influences which precursor end is diced and whether 24 or 23 nt siRNAs are produced. By diversifying the size, sequence, and strand specificity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow for maximal siRNA coverage at methylated target loci.

Biophysical characterization of Entamoeba histolytica phosphoserine aminotransferase (EhPSAT): role of cofactor and domains in stability and subunit assembly.

We investigated the role of the cofactor PLP and its binding domain in stability and subunit assembly of phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. Presence of cofactor influences the tertiary structure of EhPSAT because of the significant differences in the tryptophan microenvironment and proteolytic pattern of holo- and apo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. Stability of the protein is significantly affected by the cofactor as holo-enzyme shows higher T(m) and C(m) values for thermal and GdnHCl-induced denaturation, respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although urea-dependent unfolding of both holo- and apo-EhPSAT is a three-state process, the intermediates stabilized during unfolding are significantly different. For holo-EhPSAT a dimeric holo-intermediate was stabilized, whereas for apo-EhPSAT, a monomeric intermediate was stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer, thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild-type enzyme. To the best of our knowledge, this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.

A Comprehensive Guide to the Commercial Baculovirus Expression Vector Systems for Recombinant Protein Production.

The Baculovirus Expression Vector System (BEVS) is a workhorse for recombinant protein expression for over thirty-five years. Ever since it was first used to overexpress the human IFN-ß protein, the system has been engineered and modified several times for quick and easy expression and scale-up of the recombinant proteins. Multiple gene assemblies performed on the baculovirus genome using synthetic biology methods lead to optimized overexpression of the multiprotein complexes. Nowadays, several commercially available BEVS platforms offer a variety of customizable features, and often it is confusing which one to choose for a novice user. This short review is intended to be a one-stop guide to the commercially available baculovirus technology for heterologous protein expression in the insect cells, which users can refer to choose from popular and desirable BEVS products or services.

Affinity Tags for Protein Purification.

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

New insight on 8-anilino-1-naphthalene sulfonic acid interaction with TgFNR for hydrophobic exposure analysis.

The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.

Characterization of pH-induced transitions of Entamoeba histolytica D-phosphoglycerate dehydrogenase.

Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a functionally active homodimer at pH 7. Our earlier studies have shown that ionic interactions are essentially required for the oligomeric status and activity of the protein. Present study focuses on pH associated structural modulations of EhPGDH. Far-UV CD spectra showed loss in the secondary structure of the protein as a function of low pH, however, the protein was not completely unfolded even at pH 2. Energy minimized average simulated models of EhPGDH at different pH show stable secondary structure elements in the nucleotide binding domain (NBD) however, the substrate binding domain (SBD) was more sensitive toward acidic pH and completely unfolds at pH 2. The data suggest presence of partially folded/unfolded intermediate state at pH 2. Size exclusion chromatography shows that this intermediate has larger hydrodynamic radius compared with dimer (pH 7) or monomer (pH 5). The intermediate has poor tertiary organization with significantly exposed hydrophobic patches monitored by pH-dependent fluorescence spectroscopy and molecular dynamic simulations. Collectively, the results suggest that the two domains (NBD and SBD) of EhPGDH have independent pH-dependent structural transitions with stabilization of an intermediate state at pH 2.

Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis.

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3' termini. The 24 nt siRNAs primarily correspond to the 5' or 3' ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

Novel protein-protein interactions between Entamoeba histolyticad-phosphoglycerate dehydrogenase and phosphoserine aminotransferase.

Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein-protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10(-7) M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex.

Glu-108 is essential for subunit assembly and dimer stability of D-phosphoglycerate dehydrogenase from Entamoeba histolytica.

D-phosphoglycerate dehydrogenase catalyses the first step of phosphorylated serine biosynthesis pathway by oxidizing 3-phosphoglycerate, a glycolysis intermediate into phosphohydroxsy pyruvate. For Entamoeba histolytica this pathway is an integral part of the cysteine metabolism, which is considered to be vital for growth and survival of the parasite. Entamoeba histolytica D-phosphoglycerate dehydrogenase (EhPGDH) exists as a homodimer at pH 7. Mild acidic conditions induce significant changes in the functional and structural features of the protein as observed by enzymatic activity, spectropolarimetric measurements and fluorescence spectroscopy. Most interestingly the oligomeric status of the protein was lost and a functionally inactive monomer was stabilized at pH 5. Computational modeling and molecular dynamic simulations show that dimeric assembly of EhPGDH was stabilized with the help of several inter-subunit non-covalent interactions and subunit dissociation at pH 5 can be attributed to protonation of acidic amino acid residues present at the dimer interface. Site directed mutagenesis studies suggest that Glu-108 is essential for subunit assembly as the E108A mutant existed as monomer even at pH 7. The studies unequivocally show that the electrostatic interactions at the dimer interface play a crucial role in the stability of the protein and a complete dimer is essentially required for optimal enzymatic activity.

Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship.

BACKGROUND: Presence of phosphorylated Serine biosynthesis pathway upstream to the de novo cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by cysteine synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme. FINDINGS: EhPSAT is a homodimer of molecular mass 86 kDa. To understand the structural modulations associated with pH dependent changes in functional activity of EhPSAT detailed biophysical studies were carried out. pH alterations had no significant effect on the secondary structure, cofactor orientation and oligomeric configuration of the enzyme however, pH dependent compaction in molecular dimensions was observed. Most interestingly, a direct correlation between pH induced modulation of functional activity and orientation of Trp 101 present in the active site of the enzyme was observed. Sodium halides nullified the pH induced global changes in the enzyme, however differential effect of these salts on the active site microenvironment and functional activity of the enzyme was observed. CONCLUSIONS: The study unequivocally demonstrates that pH induced selective modification of active site microenvironment and not global change in structure or oligomeric status of the enzyme is responsible for the pH dependent change in enzymatic activity of PSAT.