Andrei Alexandrov

A Facile Method for High-throughput Co-expression of Protein Pairs

We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61-nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids. Tandem plasmids are formed in a ligation-independent manner, propagate efficiently, and produce protein pairs in high quantities. This greatly facilitates co-expression for structural genomics projects that produce thousands of clones bearing identical origins and antibiotic markers.

Determination of the oxidation states of manganese in brain, liver, and heart mitochondria

Excess brain manganese can produce toxicity with symptoms that resemble those of Parkinsonism and causes that remain elusive. Manganese accumulates in mitochondria, a major source of superoxide, which can oxidize Mn2+ to the powerful oxidizing agent Mn3+. Oxidation of important cell components by Mn3+ has been suggested as a cause of the toxic effects of manganese. Determining the oxidation states of intramitochondrial manganese could help to identify the dominant mechanism of manganese toxicity. Using X‐ray absorbance near edge structure (XANES) spectroscopy, we have characterized the oxidation state of manganese in mitochondria isolated from brain, liver, and heart over concentrations ranging from physiological to pathological. Results showed that (i) spectra from different model manganese complexes of the same oxidation state were similar to each other and different from those of other oxidation states and that the position of the absorption edge increases with oxidation state; (ii) spectra from intramitochondrial manganese in isolated brain, heart and liver mitochondria were virtually identical; and (iii) under these conditions intramitochondrial manganese exists primarily as a combination of Mn2+ complexes. No evidence for Mn3+ was detected in samples containing more than endogenous manganese levels, even after incubation under conditions promoting reactive oxygen species (ROS) production. While the presence of Mn3+ complexes cannot be proven in the spectrum of endogenous mitochondrial manganese, the shape of this spectrum could suggest the presence of Mn3+ near the limit of detection, probably as MnSOD.

Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism

Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (Ψ35, Ψ42, and Ψ44). Pus7p and Pus1p catalyze the formation of Ψ35 and Ψ44, respectively, but the mechanism of Ψ42 formation remains unclear. Using a U2 substrate containing a single 32P radiolabel at position 42, we screened a GST‐ORF library for pseudouridylase activity. Surprisingly, we found a Ψ42‐specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP (small nucleolar/Cajal body‐specific ribonucleoprotein). When isolated by tandem affinity purification (TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease‐treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the Ψ42‐specific guide RNA. Using the snR81‐deletion strain, Nhp2p‐ or Cbf5p‐conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA‐dependent and RNA‐independent mechanisms.

pH-induced folding of an apoptotic coiled coil

Par‐4 is a 38‐kD protein pivotal to the apoptotic pathways of various cell types, most notably prostate cells and neurons, where it has been linked to prostate cancer and various neurodegenerative disorders including Alzheimers and Huntingtons diseases and HIV encephalitis. The C‐terminal region of Par‐4 is responsible for homodimerization and the ability of Par‐4 to interact with proposed effector molecules. In this study, we show that the C‐terminal 47 residues of Par‐4 are natively unfolded at physiological pH and temperature. Evidence is rapidly accumulating that natively unfolded proteins play an important role in various cellular functions and signaling pathways, and that folding can often be induced on complexation with effector molecules or alteration of environment. Here we use primarily CD studies to show that changes in the environment, particularly pH and temperature, can induce the Par‐4 C terminus to form a self‐associated coiled coil.

Phosphorylation of DGCR8 Increases Its Intracellular Stability and Induces a Progrowth miRNA Profile

SUMMARY During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.

Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay

The multiprotein exon junction complex (EJC) that is deposited upstream of spliced junctions orchestrates downstream events in the life of a metazoan mRNA, including its surveillance via the nonsense-mediated decay (NMD) pathway. However, the mechanism by which the spliceosome mediates EJC formation is not well understood. We show that human eIF4G-like spliceosomal protein (h)CWC22 directly interacts with the core EJC component eIF4AIII in vitro and in vivo; mutations at the predicted hCWC22/eIF4AIII interface disrupt association. In vivo depletion of hCWC22, as for yeast Cwc22p, causes a splicing defect, resulting in decreased levels of mature cellular mRNAs. Nonetheless, hCWC22 depletion yields increased levels of spliced RNA from the unusual nonsense codon-containing U22 host gene, which is a natural substrate of NMD. To test whether hCWC22 acts in NMD through coupling splicing to EJC deposition, we searched for mutations in hCWC22 that affect eIF4AIII deposition without affecting splicing. Addition of hCWC22(G168Y) with a mutation at the putative hCWC22/eIF4AIII interface exacerbates the defect in splicing-dependent deposition of eIF4AIII(T334V) with a mutation reported to be in direct contact with mRNA, linking hCWC22 to the process of EJC deposition in vitro. Importantly, the addition of hCWC22(G168Y) affects deposition of eIF4AIII(T334V) without inhibiting splicing or the efficiency of deposition of the endogenous eF4AIII(WT) in the same reaction, demonstrating hCWC22’s specific role in eIF4AIII deposition in addition to its role in splicing. The essential splicing factor CWC22 has, therefore, acquired functions in EJC assembly and NMD during evolution from single-celled to complex eukaryotes.

Human eIF4AIII interacts with an eIF4G-like partner, NOM1, revealing an evolutionarily conserved function outside the exon junction complex

Despite the lack of an exon junction complex (EJC), Saccharomyces cerevisiae contains Fal1p, a DEAD-box helicase highly homologous to eIF4AIII. We show that yeast Fal1p is functionally orthologous to human eIF4AIII, since expression of human eIF4AIII complements both the lethal phenotype and the 18S rRNA biogenesis defect of fal1Δ(null) yeast. We further show that yeast Fal1p interacts genetically with an eIF4G-like protein, Sgd1p: One allele of sgd1 acts as a dominant extragenic suppressor of a mutation in a predicted RNA-binding residue of Fal1p, whereas another synthetically exacerbates the growth defect of this fal1 mutation. Both sgd1 mutations map to a single, short, evolutionarily conserved patch that matches key eIF4A-interacting residues of eIF4G when superimposed on the X-ray structure of the eIF4A/eIF4G complex. We demonstrate direct physical interactions between yeast Sgd1p and Fal1p, and between their human orthologs (NOM1 and eIF4AIII) in vitro and in vivo, identifying human NOM1 as a missing eIF4G-like interacting partner of eIF4AIII. Knockdown of eIF4AIII and NOM1 in human cells demonstrates that this novel conserved eIF4A/eIF4G-like complex acts in pre-rRNA processing, adding to the established functions of eIF4A/eIF4G in translation initiation and of eIF4AIII as the core component of the EJC.

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding.

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.

The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells

A substrate for protein kinase B (PKB)α in HeLa cell extracts was identified as methyltransferase‐like protein‐1 (METTL1), the orthologue of trm8, which catalyses the 7‐methylguanosine modification of tRNA in Saccharomyces cerevisiae. PKB and ribosomal S6 kinase (RSK) both phosphorylated METTL1 at Ser27 in vitro. Ser27 became phosphorylated when HEK293 cells were stimulated with insulin‐like growth factor‐1 (IGF‐1) and this was prevented by inhibition of phosphatidyinositol 3‐kinase. The IGF‐1‐induced Ser27 phosphorylation did not occur in 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1)‐deficient embryonic stem cells, but occurred normally in PDK1[L155E] cells, indicating that the effect of IGF‐1 is mediated by PKB. METTL1 also became phosphorylated at Ser27 in response to phorbol‐12‐myristate 13‐acetate and this was prevented by PD 184352 or pharmacological inhibition of RSK. Phosphorylation of METTL1 by PKB or RSK inactivated METTL1 in vitro, as did mutation of Ser27 to Asp or Glu. Expression of METTL1[S27D] or METTL1[S27E] did not rescue the growth phenotype of yeast lacking trm8. In contrast, expression of METTL1 or METTL1[S27A] partially rescued growth. These results demonstrate that METTL1 is inactivated by PKB and RSK in cells, and the potential implications of this finding are discussed.

Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations

The apoptosis‐associated Par‐4 protein has been implicated in cancers of the prostate, colon, and kidney, and in Alzheimers and Huntingtons diseases, among other neurodegenerative disorders. Previously, we have shown that a peptide from the Par‐4 C‐terminus, which is responsible for Par‐4 self‐association as well as interaction with all currently identified effector molecules, is natively unfolded at neutral pH, but forms a tightly associated coiled coil at acidic pH and low temperature. Here, we have alternately mutated the two acidic residues predicted to participate in repulsive electrostatic interactions at the coiled coil interhelical interface. Analysis of circular dichroism spectra reveals that a dramatic alteration of the folding/unfolding equilibrium of this peptide can be effected through directed‐point mutagenesis, confirming that the two acidic residues are indeed key to the pH‐dependent folding behavior of the Par‐4 coiled coil, and further suggesting that alleviation of charge repulsion through exposure to either a low pH microenvironment or an electrostatically complementary environment may be necessary for efficient folding of the Par‐4 C‐terminus.