Nina Reuven

The Mutagenesis Protein UmuC Is a DNA Polymerase Activated by UmuD′, RecA, and SSB and Is Specialized for Translesion Replication

Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD′. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191–199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD′, RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD′, RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10–100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD′-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the humanXP-V genes, encoding DNA polymerase η.

Yap1 Phosphorylation by c-Abl Is a Critical Step in Selective Activation of Proapoptotic Genes in Response to DNA Damage

Cells undergo apoptosis upon exposure to severe DNA damage stress. Under this condition, p73 is phosphorylated and activated by c-Abl. The transcription coactivator Yap1 binds p73 to generate a complex that escapes p73 proteasomal degradation and recruits p300 to support transcription of proapoptotic genes. However, the mechanism of selective activation of proapoptotic genes by Yap1 remained unclear. In this study, we show that c-Abl directly phosphorylates Yap1 at position Y357 in response to DNA damage. Tyrosine-phosphorylated Yap1 is a more stable protein that displays higher affinity to p73 and selectively coactivates p73 proapoptotic target genes. Furthermore, we show that Yap1 switches between p73-mediated proapoptotic and growth arrest target genes based on its phosphorylation state. Thus, our data demonstrate that modification of a transcription coactivator, namely the DNA damage-induced phosphorylation of Yap1 by c-Abl, influences the specificity of target gene activation.

The Yes-associated protein 1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73

Upon DNA damage signaling, p73, a member of the p53 tumor suppressor family, accumulates to support transcription of downstream apoptotic genes. p73 interacts with Yes-associated protein 1 (Yap1) through its PPPY motif, and increases p73 transactivation of apoptotic genes. The ubiquitin E3 ligase Itch, like Yap1, interacts with p73. Given the fact that both Itch and Yap1 bind p73 via the PPPY motif, we hypothesized that Yap may also function to stabilize p73 by displacing Itch binding to p73. We show that the interaction of Yap1 and p73 was necessary for p73 stabilization. Yap1 competed with Itch for binding to p73, and prevented Itch-mediated ubiquitination of p73. Treatment of cells with cisplatin leads to an increase in p73 accumulation and induction of apoptosis, but both were dramatically reduced in the presence of Yap1 siRNA. Altogether, our findings attribute a central role to Yap1 in regulating p73 accumulation and function under DNA damage signaling.

Ubiquitin-independent p53 proteasomal degradation

The mechanism of p53 proteasomal degradation through polyubiquitination is well characterized. The basic assumption behind this mechanism is that p53 is inherently stable unless sensitized to degradation by polyubiquitination. However, a number of studies provide evidence for p53 to be naturally unstable. Consistent with this attribute is the fact that both p53 N- and C-termini are intrinsically unstructured. Recent findings provide evidence for p53 to be degraded by the 20S proteasome by default unless it escapes this process. A number of mechanisms were demonstrated and proposed to play a role in rescuing p53 from default degradation. These mechanisms, their biological implications, and relevance to cancer are reviewed in this article.

Operational definition of intrinsically unstructured protein sequences based on susceptibility to the 20S proteasome

Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well‐defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein–protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs. Proteins 2008.

Susceptibility of p53 Unstructured N Terminus to 20 S Proteasomal Degradation Programs the Stress Response

The N-terminal transcription activation domain of p53 is intrinsically unstructured. We show in vitro and in vivo that this domain initiates p53 degradation by the 20 S proteasome in a ubiquitin-independent fashion. The decay of metabolically labeled p53 follows biphasic kinetics with an immediate fast phase that is ubiquitin-independent and a second slower phase that is ubiquitin-dependent. The 20 S proteasome executes the first phase by default, whereas the second phase requires the 26 S proteasome. p53 N-terminal binding proteins, such as Hdmx, can selectively block the first phase of degradation. Remarkably, γ-irradiation inhibits both p53 decay phases, whereas UV selectively negates the second phase, giving rise to discrete levels of p53 accumulation. Our data of a single protein experiencing double mode degradation mechanisms each with unique kinetics provide the mechanistic basis for programmable protein homeostasis (proteostasis).

Autorepression of Rfx1 Gene Expression: Functional Conservation from Yeast to Humans in Response to DNA Replication Arrest

ABSTRACT The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.

The protein level of PGC-1α, a key metabolic regulator, is controlled by NADH-NQO1

ABSTRACT PGC-1α is a key transcription coactivator regulating energy metabolism in a tissue-specific manner. PGC-1α expression is tightly regulated, it is a highly labile protein, and it interacts with various proteins—the known attributes of intrinsically disordered proteins (IDPs). In this study, we characterize PGC-1α as an IDP and demonstrate that it is susceptible to 20S proteasomal degradation by default. We further demonstrate that PGC-1α degradation is inhibited by NQO1, a 20S gatekeeper protein. NQO1 binds and protects PGC-1α from degradation in an NADH-dependent manner. Using different cellular physiological settings, we also demonstrate that NQO1-mediated PGC-1α protection plays an important role in controlling both basal and physiologically induced PGC-1α protein level and activity. Our findings link NQO1, a cellular redox sensor, to the metabolite-sensing network that tunes PGC-1α expression and activity in regulating energy metabolism.

A Regulatory Circuit Controlling Itch-mediated p73 Degradation by Runx

The members of the tumor suppressor p53 family are under tight regulation by distinct ubiquitin-protein isopeptide (E3) ligases. The level of p73 is regulated by the E3 ligase Itch. Itch levels are sharply reduced in response to DNA damage with concomitant p73 accumulation and activation. The mechanism of controlling Itch level is not known. We show that the Itch promoter is a target of the transcription activator Runx. Yes-associated protein (Yap1) is a shared transcription co-activator of Runx and p73. Under normal conditions, the Runx-Yap1 complex binds the Itch promoter and supports its transcription and p73 degradation. In response to DNA damage, Yap1 is phosphorylated by c-Abl at the position Tyr-357. The modified Yap1 does not co-activate Runx in supporting Itch transcription. The subsequent reduction in the Itch level gives rise to p73 accumulation. These results demonstrate how Yap1 supports degradation of p73 via Runx and how it plays an opposite role in response to DNA damage.

c-Fos Proteasomal Degradation Is Activated by a Default Mechanism, and Its Regulation by NAD(P)H:Quinone Oxidoreductase 1 Determines c-Fos Serum Response Kinetics

ABSTRACT The short-lived proto-oncoprotein c-Fos is a component of the activator protein 1 (AP-1) transcription factor. A large region of c-Fos is intrinsically unstructured and susceptible to a recently characterized proteasomal ubiquitin-independent degradation (UID) pathway. UID is active by a default mechanism that is inhibited by NAD(P)H:quinone oxidoreductase 1 (NQO1), a 20S proteasome gatekeeper. Here, we show that NQO1 binds and induces robust c-Fos accumulation by blocking the UID pathway. c-Jun, a partner of c-Fos, also protects c-Fos from proteasomal degradation by default. Our findings suggest that NQO1 protects monomeric c-Fos from proteasomal UID, a function that is fulfilled later by c-Jun. We show that this process regulates c-Fos homeostasis (proteostasis) in response to serum stimulation, phosphorylation, nuclear translocation, and transcription activity. In addition, we show that NQO1 is important to ensure immediate c-Fos accumulation in response to serum, since a delayed response was observed under low NQO1 expression. These data suggest that in vivo, protein unstructured regions determine the kinetics and the homeostasis of regulatory proteins. Our data provide evidence for another layer of regulation of key regulatory proteins that functions at the level of protein degradation and is designed to ensure optimal formation of functional complexes such as AP-1.