Chin Ha Chung

Transcriptional regulation of a metastasis suppressor gene by Tip60 and |[beta]|-catenin complexes

Defining the molecular strategies that integrate diverse signalling pathways in the expression of specific gene programmes that are critical in homeostasis and disease remains a central issue in biology. This is particularly pertinent in cancer biology because downregulation of tumour metastasis suppressor genes is a common occurrence, and the underlying molecular mechanisms are not well established. Here we report that the downregulation of a metastasis suppressor gene, KAI1, in prostate cancer cells involves the inhibitory actions of β-catenin, along with a reptin chromatin remodelling complex. This inhibitory function of β-catenin–reptin requires both increased β-catenin expression and recruitment of histone deacetylase activity. The coordinated actions of β-catenin–reptin components that mediate the repressive state serve to antagonize a Tip60 coactivator complex that is required for activation; the balance of these opposing complexes controls the expression of KAI1 and metastatic potential. The molecular mechanisms underlying the antagonistic regulation of β-catenin–reptin and the Tip60 coactivator complexes for the metastasis suppressor gene, KAI1, are likely to be prototypic of a selective downregulation strategy for many genes, including a subset of NF-κB target genes.

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

Primary structures of two homologous subunits of PA28, a γ-interferon-inducible protein activator of the 20S proteasome

The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28α and PA28β, are about 50% identical to one another and are highly conserved between rat and human. PA28α and PA28β are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28α, but neither PA28β nor Ki antigen, contains a ‘KEKE motif’, which has been postulated to promote the binding of proteins having this structural feature. PA28α and PA28β were coordinately regulated by γ‐interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to γ‐interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.

Nucleotide-Dependent Conformational Changes in a Protease-Associated ATPase HslU

BACKGROUND The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

Versatile protein tag, SUMO: its enzymology and biological function.

Small ubiquitin‐related modifier (SUMO) is a member of a ubiquitin‐like protein family that regulates cellular function of a variety of target proteins. SUMO and ubiquitin are synthesized as precursors that need to be processed prior to conjugation to target proteins, and their mature forms have a similar tertiary structure. The mechanism for SUMO conjugation is also analogous to that of the ubiquitin system, such as the utilization of E1, E2, and E3 cascade enzymes. However, the biological consequence of SUMO modification is quite different from that of the ubiquitin system. Whereas ubiquitination of most proteins is for the degradative pathway, SUMO modification of target proteins is involved in nuclear protein targeting, formation of subnuclear structures, regulation of transcriptional activities or DNA binding abilities of transcription factors, and control of protein stability. This review will summarize the recent progress made in the enzymology of SUMO and its biological significance.

Dual Modification of BMAL1 by SUMO2/3 and Ubiquitin Promotes Circadian Activation of the CLOCK/BMAL1 Complex

ABSTRACT Heterodimers of BMAL1 and CLOCK drive rhythmic expression of clock-controlled genes, thereby generating circadian physiology and behavior. Posttranslational modifications of BMAL1 play a key role in modulating the transcriptional activity of the CLOCK/BMAL1 complex during the circadian cycle. Recently, we demonstrated that circadian activation of the heterodimeric transcription factor is accompanied by ubiquitin-dependent proteolysis of BMAL1. Here we show that modification by SUMO localizes BMAL1 exclusively to the promyelocytic leukemia nuclear body (NB) and simultaneously promotes its transactivation and ubiquitin-dependent degradation. Under physiological conditions, BMAL1 was predominantly conjugated to poly-SUMO2/3 rather than SUMO1, and the level of these conjugates underwent rhythmic variation, peaking at times of maximum E-box-mediated circadian transcription. Interestingly, mutation of the sumoylation site (Lys259) of BMAL1 markedly inhibited both its ubiquitination and its proteasome-mediated proteolysis, and these effects were reversed by covalent attachment of SUMO3 to the C terminus of the mutant BMAL1. Consistent with this, SUSP1, a SUMO protease highly specific for SUMO2/3, abolished ubiquitination, as well as sumoylation of BMAL1, while the ubiquitin protease UBP41 blocked BMAL1 ubiquitination but induced accumulation of polysumoylated BMAL1 and its localization to the NB. Furthermore, inhibition of proteasome with MG132 elicited robust nuclear accumulation of SUMO2/3- and ubiquitin-modified BMAL1 that was restricted to the transcriptionally active stage of the circadian cycle. These results indicate that dual modification of BMAL1 by SUMO2/3 and ubiquitin is essential for circadian activation and degradation of the CLOCK/BMAL1 complex.

Roles of sumoylation of a reptin chromatin-remodelling complex in cancer metastasis

Defining the functional modules within transcriptional regulatory factors that govern switching between repression and activation events is a central issue in biology. Recently, we have reported the dynamic role of a β-catenin–reptin chromatin remodelling complex in regulating a metastasis suppressor gene KAI1 (ref.1), which is capable of inhibiting the progression of tumour metastasis. Here, we identify signalling factors that confer repressive function on reptin and hence repress the expression of KAI1. Biochemical purification of a reptin-containing complex has revealed the presence of specific desumoylating enzymes that reverse the sumoylation of reptin that underlies its function as a repressor. Desumoylation of reptin alters the repressive function of reptin and its association with HDAC1. Furthermore, the sumoylation status of reptin modulates the invasive activity of cancer cells with metastatic potential. These data clearly define a functional model and provide a novel link for SUMO modification in cancer metastasis.

BTB Domain-containing Speckle-type POZ Protein (SPOP) Serves as an Adaptor of Daxx for Ubiquitination by Cul3-based Ubiquitin Ligase

Daxx is a multifunctional protein that regulates a variety of cellular processes, including transcription, cell cycle, and apoptosis. SPOP is a BTB (Bric-a-brac/Tramtrack/Broad complex) protein that constitutes Cul3-based ubiquitin ligases. Here we show that SPOP serves as an adaptor of Daxx for the ubiquitination by Cul3-based ubiquitin ligase and subsequent degradation by the proteasome. Expression of SPOP with Cul3 markedly reduced Daxx level, and this degradation was blocked by SPOP-specific short hairpin RNAs. Inhibition of the proteasome by MG132 caused the prevention of Daxx degradation in parallel with the accumulation of ubiquitinated Daxx. Expression of SPOP with Cul3 reversed Daxx-mediated repression of ETS1- and p53-dependent transcription, and short hairpin RNA-mediated knock down of SPOP blocked the recovery of their transcriptional activation. Furthermore, Daxx degradation led to the cleavage of poly(ADP-ribose) polymerase and the increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end-labeling-positive apoptotic cells. These results suggest that SPOP/Cul3-ubiquitin ligase plays an essential role in the control of Daxx level and, thus, in the regulation of Daxx-mediated cellular processes, including transcriptional regulation and apoptosis.

Molecular properties of the proteasome activator PA28 family proteins and γ-interferon regulation

Background: Recent cDNA cloning of two homologous proteasome activators, PA28α and PA28β, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I‐restricted antigen processing pathway. Recently, an immunomodulatory cytokine γ‐interferon (γ‐IFN) was found to increase greatly the messages for PA28α and PA28β, but not Ki antigen, in human cells.

ISG15 and immune diseases

Abstract ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. ISG15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the C-terminal glycine residue before conjugation to target proteins. A set of three-step cascade enzymes, an E1 enzyme (UBE1L), an E2 enzyme (UbcH8), and one of several E3 ligases (e.g., EFP and HERC5), catalyzes ISG15 conjugation (ISGylation) of a specific protein. These enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide. Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15. Some of them are type I IFN-induced proteins, such as PKR and RIG-I, and some are the key regulators that are involved in IFN signaling, such as JAK1 and STAT1, implicating the role of ISG15 and its conjugates in type I IFN-mediated innate immune responses. However, relatively little is known about the functional significance of ISG15 induction due to the lack of information on the consequences of its conjugation to target proteins. Here, we describe the recent progress made in exploring the biological function of ISG15 and its reversible modification of target proteins and thus in their implication in immune diseases.