SUMMARYThe literature review presented here details recent research involving members of the poly(ADP-ribose) polymerase (PARP) family of proteins. Among the 17 recognized members of the family, the human enzyme PARP1 is the most extensively studied, resulting in a number of known biological and metabolic roles. This review is focused on the roles played by PARP enzymes in host-pathogen interactions and in diseases with an associated inflammatory response. In mammalian cells, several PARPs have specific roles in the antiviral response; this is perhaps best illustrated by PARP13, also termed the zinc finger antiviral protein (ZAP). Plant stress responses and immunity are also regulated by poly(ADP-ribosyl)ation. PARPs promote inflammatory responses by stimulating proinflammatory signal transduction pathways that lead to the expression of cytokines and cell adhesion molecules. Hence, PARP inhibitors show promise in the treatment of inflammatory disorders and conditions with an inflammatory component, such as diabetes, arthritis, and stroke. These functions are correlated with the biophysical characteristics of PARP family enzymes. This work is important in providing a comprehensive understanding of the molecular basis of pathogenesis and host responses, as well as in the identification of inhibitors. This is important because the identification of inhibitors has been shown to be effective in arresting the progression of disease.
Hyperthermophilic archaeal viruses, including Sulfolobus spindle-shaped viruses (SSVs) such as SSV-1 and SSV-Ragged Hills, exhibit remarkable morphology and genetic diversity. However, they remain poorly understood, in part because their genomes exhibit limited or unrecognizable sequence similarity to genes with known function. Here we report structural and functional studies of E73, a 73-residue homodimeric protein encoded within the SSV-Ragged Hills genome. Despite lacking significant sequence similarity, the nuclear magnetic resonance (NMR) structure reveals clear similarity to ribbon–helix–helix (RHH) domains present in numerous proteins involved in transcriptional regulation. In vitro double-stranded DNA (dsDNA) binding experiments confirm the ability of E73 to bind dsDNA in a nonspecific manner with micromolar affinity, and characterization of the K11E variant confirms the location of the predicted DNA binding surface. E73 is distinct, however, from known RHH domains. The RHH motif is elaborated upon by the insertion of a third helix that is tightly integrated into the structural domain, giving rise to the “RH3” fold. Within the homodimer, this helix results in the formation of a conserved, symmetric cleft distal to the DNA binding surface, where it may mediate protein–protein interactions or contribute to the high thermal stability of E73. Analysis of backbone amide dynamics by NMR provides evidence of a rigid core, fast picosecond to nanosecond time scale NH bond vector motions for residues located within the antiparallel β-sheet region of the proposed DNA-binding surface, and slower microsecond to millisecond time scale motions for residues in the α1−α2 loop. The roles of E73 and its SSV homologues in the viral life cycle are discussed.
Backbone amide dynamics studies were conducted on a temperature-sensitive mutant (L75F-TrpR) of the tryptophan repressor protein (TrpR) of Escherichia coli in its apo (i.e., no l-tryptophan corepressor-bound) form. The (15)N NMR relaxation profiles of apo-L75F-TrpR were analyzed and compared to those of wild-type (WT) and super-repressor mutant (A77V) TrpR proteins, also in their apo forms. The (15)N NMR relaxation data ((15)N-T(1), (15)N-T(2), and heteronuclear (15)N-{(1)H}-nOe) recorded on all three aporepressors at a magnetic field strength of 600 MHz ((1)H Larmor frequency) were analyzed to extract dynamics parameters, including diffusion tensor ratios (D(∥)/D(⊥)), correlation times (τ(m)) for overall reorientations of the proteins in solution, reduced spectral density terms [J(eff)(0), J(0.87ω(H)), J(ω(N))], and generalized order parameters (S(2)), which report on protein internal motions on the picosecond to nanosecond and slower microsecond to millisecond chemical exchange time scales. Our results indicate that all three aporepressors exhibit comparable D(∥)/D(⊥) ratios and characteristic time constants, τ(m), for overall global reorientation, indicating that in solution, all three apoproteins display very similar overall shape, structure, and rotational diffusion properties. Comparison of (15)N NMR relaxation data, reduced spectral density profiles, and generalized S(2) order parameters indicated that these parameters are quite uniform for backbone amides positioned within the four (A-C and F) core α-helices of all three aporepressors. In contrast, small but noticeable differences in internal dynamics were observed for backbone amides located within the helix D-turn-helix E DNA-binding domain of the apo-TrpR proteins. The significance of these dynamics differences in terms of the biophysical characteristics and ligand binding properties of the three apo-TrpR proteins is discussed.
Backbone amide dynamics of the Escherichia coli tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo-(L-tryptophan (L-Trp) co-repressor-bound) form have been characterized using 15N NMR relaxation. The three proteins possess very similar structures ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific 15N-T1, 15N-T2, 15N-{1H}-nOes, reduced spectral density, and generalized order parameters (S2), indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins’ DNA-binding domain. We find that flexibility is highly restricted for amides in core α-helices (i.e. helices A, B, C, and F), and a comparable “stiffening” is observed for residues in the DNA recognition helix (helix E) of the helix-D-turn-helix-E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA-binding, and molecular recognition of target operators. Comparison of the 15N NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicate that the single point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways, and are most pronounced in the apo-forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.
Lines 2 to 6 should read ".. . PARP12 shares some of the observed roles of PARP13 in RNA decay (36) and in the antiviral response. Like PARP13, it recognizes specific sequences in viral RNA and DNA (121, 122, 125, 137). Both proteins contain zinc finger domains in the N-terminal region. Recognition of RNA by these nucleic acid-binding domains is required for antiviral activity (129, 130, 137)." Page 7, paragraph 6: Lines 1 to 7 should read "PARP7,-10,-12, and-13 are all induced by interferons, and all can inhibit viral replication (135, 136). PARP7 and PARP10, which are MARTs, are capable of translation inhibition. These proteins form complexes with ribosomes that are mediated by their N-terminal RNA-binding domains (137). Translation inhibition prevents viral growth by stopping viral protein synthesis. The finding that the induction of PARP7,-10,-12, and-13 can result in the inhibition of virus replication is consistent with previous results supporting the importance of PARPs in the inhibition of viral replication (135, 136)." Page 9, paragraph 1: Lines 8 to 10 should read ".. . The RNA-binding PARP12 and-13 are found in stress granules, where PARPs are responsible for MARylation of various proteins such as the argonaute proteins, G3BP1, and TIA-1 (36)."
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