Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase.
Jenna Hendershot, with her mentor Professor Patrick O'Brien, and recent graduate Dr. Abigail Wolfe collaborated on a paper for Biochemistry and had some surprising results.
Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs. PubMed
Hendershot JM, Wolfe AE, O'Brien PJ.
Biochemistry. 2011 Mar 22;50(11):1864-74. Epub 2011 Feb 3.
Professor Alex Ninfa and Peng Jiang received notice of this work in the Editor's Choice column of the October 21 issue of Science.
Biological signal transduction networks are commonly viewed as circuits that pass along information--in the process amplifying signals, enhancing sensitivity, or performing other signal-processing tasks--to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a circuit's ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets. PubMed
Jiang P, Ninfa AJ
Biochemistry. 2011 Nov 15. [Epub ahead of print]
A University of Michigan and University of Oxford collaboration recently earned front page attention in the Royal Society of Chemistry’s journal
Read more on the team’s work, including its earlier coverage in the Journal of the American Chemical Society.
Alex Ninfa and Peng Jiang received notice in the Editor's Choice column of Science for their collaboration "Load-induced modulation of signal transduction networks," published on October 11. Jiang and Ninfa explored the concept of “load” by applying it to biochemical signaling systems; that is, whether the dynamic properties of a signaling mechanism were altered in the presence or absence of substrate molecules that are targets of the system. Combined experiments and mathematical modeling showed that the presence of substrate could alter the response time of the system, increasing it when one of the enzymes in the signaling system was operating at a maximal rate (saturated) but decreasing it when the enzymes were operating in a linear manner. The authors discuss how such effects of downstream targets on the responsiveness of signaling systems might be used to design appropriate responses when modifying biological systems or designing synthetic ones. Read the abstract here.
Professors Jason Gestwicki and Georgios Skiniotis have collaborated with graduate students in the ChemBio grad program and published Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK) in a recent isssue of Cell Stress Chaperones. Their studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling. Read the abstract here PMID: 22076723
Trievel Lab: Structural and Functional Analysis of JMJD2D Reveals Molecular Basis for Site-Specific Demethylation among JMJD2 Demethylases
JMJD2 lysine demethylases (KDMs) participate in diverse genomic processes. Most JMJD2 homologs display dual selectivity toward H3K9me3 and H3K36me3, with the exception of JMJD2D, which is specific for H3K9me3. Swathi Krishnan and Raymond C. Trievel report onthe crystal structures of the JMJD2D⋅2-oxoglutarate⋅H3K9me3 ternary complex and JMJD2D apoenzyme. In a recent paper published in Structure. Utilizing structural alignments with JMJD2A, molecular docking, and kinetic analysis with an array of histone peptide substrates, we elucidate the specific signatures that permit efficient recognition of H3K9me3 by JMJD2A and JMJD2D, and the residues in JMJD2D that occlude H3K36me3 demethylation. Surprisingly, these results reveal that JMJD2A and JMJD2D exhibit subtle yet important differences in H3K9me3 recognition, despite the overall similarity in the substrate-binding conformation. Further, we show that H3T11 phosphorylation abrogates demethylation by JMJD2 KDMs. Together, these studies reveal the molecular basis for JMJD2 site specificity and provide a framework for structure-based design of selective inhibitors of JMJD2 KDMs implicated in disease. More
Krishnan S and Trievel RC
Structure, Vol. 21, Issue 1, 8 January 2013, Pages 98–108
Dou Lab: High-Affinity, Small-Molecule Peptidomimetic Inhibitors of MLL1/WDR5 Protein-Protein Interaction
Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein-protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH-, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with K(i) < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein-protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.
Karatas H, Townsend EC, Cao F, Chen Y, Bernard D, Liu L, Lei M, Dou Y, Wang S.
J Am Chem Soc. 2012 Dec 27.
Banerjee Lab: Characterization of Patient Mutations in Human Persulfide Dioxygenase (ETHE1) Involved in H2S Catabolism
Hydrogen sulfide (H(2)S) is a recently described endogenously produced gaseous signaling molecule that influences various cellular processes in the central nervous system, cardiovascular system, and gastrointestinal tract. The biogenesis of H(2)S involves the cytoplasmic transsulfuration enzymes, cystathionine β-synthase and γ-cystathionase, whereas its catabolism occurs in the mitochondrion and couples to the energy-yielding electron transfer chain. Low steady-state levels of H(2)S appear to be controlled primarily by efficient oxygen-dependent catabolism via sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1), rhodanese, and sulfite oxidase. Mutations in the persulfide dioxgenase, i.e. ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared with the wild-type enzyme. The D196N mutation results in a 2-fold higher K(m) for the substrate, glutathione persulfide.
Kabil O, Banerjee R.
J Biol Chem. 2012 Dec 28;287(53):44561-7. doi: 10.1074/jbc.M112.407411.
[PubMed - in process]
[Available on 2013/12/28]
Goldstrohm Lab: Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs
PUF proteins are a conserved family of eukaryotic RNA-binding proteins that regulate specific mRNAs: they control many processes including stem cell proliferation, fertility, and memory formation. PUFs repress protein expression from their target mRNAs but the mechanism by which they do so remains unclear, especially for humans. Humans possess two PUF proteins, PUM1 and PUM2, which exhibit similar RNA binding specificities. Here we report new insights into their regulatory activities and mechanisms of action. We developed functional assays to measure sequence-specific repression by PUM1 and PUM2. Both robustly inhibit translation and promote mRNA degradation. Purified PUM complexes were found to contain subunits of the CCR4-NOT (CNOT) complex, which contains multiple enzymes that catalyze mRNA deadenylation. PUMs interact with the CNOT deadenylase subunits in vitro. We used three approaches to determine the importance of deadenylases for PUM repression. First, dominant-negative mutants of CNOT7 and CNOT8 reduced PUM repression. Second, RNA interference depletion of the deadenylases alleviated PUM repression. Third, the poly(A) tail was necessary for maximal PUM repression. These findings demonstrate a conserved mechanism of PUF-mediated repression via direct recruitment of the CCR4-POP2-NOT deadenylase leading to translational inhibition and mRNA degradation. A second, deadenylation independent mechanism was revealed by the finding that PUMs repress an mRNA that lacks a poly(A) tail. Thus, human PUMs are repressors capable of deadenylation-dependent and -independent modes of repression.
Van Etten J, Schagat TL, Hrit J, Weidmann CA, Brumbaugh J, Coon JJ, Goldstrohm AC.
J Biol Chem. 2012 Oct 19;287(43):36370-83. doi: 10.1074/jbc.M112.373522.
In Alzheimer's disease (AD) and other neurodegenerative disorders, proteins accumulate into ordered aggregates, called amyloids. Recent evidence suggests that these structures include both large, insoluble fibrils and smaller, prefibrillar structures, such as dimers, oligomers, and protofibrils. Recently, focus has shifted to the prefibrillar aggregates because they are highly neurotoxic and their levels appear to correlate with cognitive impairment. Thus, there is interest in finding methods for specifically quantifying these structures. One of the classic ways of detecting amyloid formation is through the fluorescence of the benzothiazole dye, thioflavin T (ThT). This reagent has been a "workhorse" of the amyloid field because it is robust and inexpensive. However, one of its limitations is that it does not distinguish between prefibrillar and fibrillar aggregates. BioChem's Professor Jason E. Gestwicki and Graduate Student Ashley Reinke in a recent issue of the European journal Chembiochem tell how they screened a library of 37 indoles for those that selectively change fluorescence in the presence of prefibrillar amyloid-beta (Abeta). From this process, we selected the most promising example, tryptophanol (TROL), to use in a quantitative "thioflavin-like" assay. Using this probe in combination with electron microscopy, we found that prefibrils are largely depleted during Abeta aggregation in vitro but that they remain present after the apparent saturation of the ThT signal. These results suggest that a combination of TROL and ThT provides greater insight into the process of amyloid formation by Abeta. In addition, we found that TROL also recognizes other amyloid-prone proteins, including ataxin-3, amylin, and CsgA. Thus, this assay might be an inexpensive spectroscopic method for quantifying amyloid prefibrils in vitro. The entire article may be read at Chembiochem.
The Banerjee lab, productive as always, has published Taurine biosynthesis by neurons and astrocytes, in the Journal of Biological Chemistry. Although widely known that taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies, the physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Researcher Victor Vitvitsky is first author, collaborating with Sanjay Garg and Dr. Ruma Banerjee. Read the abstract here PMID: 21778230
William E. M. Lands, who for 25 years was Professor of Biological Chemistry at the University of Michigan before becoming Senior Scientific Advisor at National Institute on Alcohol Abuse and Alcoholism, is being celebrated for his life-long work in an article in the Journal of Biological Chemistry. The Selective Placement of Acyl Chains: the Work of W.E.M. Lands
The Journal for Biological Chemistry has published Emerita Professor Rowena G. Matthews' A Love Affair with Vitamins in the July edition as part of its Reflections series. In it Matthews tells of her progress from helping her biologist father preparing beef heart mitochondria for his research, to Radcliff in the 50s, and later Ann Arbor for thesis research with Vincent Massey and postdoctoral work with Charles Williams, who with Jud Coon convinced a then cautious University to hire her on as a Assistant Professor. Matthews' subsequent adventures in biology make for compelling reading. Download the pfd.
Emeritus Professor Bernard W. Agranoff's article was published in JBC as part of its Reflections series. Starting with a year in the laboratory of Feodor Lynen, at the Max Planck Institute for Cell Chemistry in Munich, he recalls â€œparticipat[ing] in the exciting search to unravel the biosynthesis of cholesterol.â€ Later, research into the possible phosphomonoesters of myo-inositol, and the possible physiological significance, lead to some rather interesting insights in brain inositol and lithium. Agranoff ends with some speculations regarding the possibility that terrestrial inositol preceded the existence of living organisms. Download the pdf.
Despite the development of more than 25 approved anti-HIV drugs and improvements in the availability of antiretroviral drugs in low and middle income countries, the rate of new HIV-1 infections is outpacing the rate of new individuals receiving antiretroviral therapy by 2.5:1. At present, it appears that an efficacious HIV vaccine is still many years away. Therefore, other methods for halting the spread of HIV are vitally needed. This has raised the possibility of developing either intravaginally or intrarectally applied microbicides to halt the spread of HIV during sexual intercourse. This type of intervention is particularly needed in the developing world, such as sub-Saharan Africa, where more than 20 million people are living with HIV/AIDS. In a paper published in the March 19 issue of the Journal of Biological Chemistry, BioChemâ€™s Irwin Goldstein, with colleagues from UMâ€™s Division of Infectious Diseases, hypothesize that BanLec, a jacalin-related lectin isolated from the fruit of bananas, might inhibit HIV-1 through binding of the glycosylated HIV-1 envelope protein, gp120. For more on this exciting research, read the full paper at JBC