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Cks proteins are small evolutionarily conserved proteins that interact genetically and physically with cyclin-dependent kinases. However, in spite of a large body of genetic, biochemical and structural research, no compelling unifying model of their functions has emerged 1 , 2 . Here we show, by investigating the essential role of Cks1 in Saccharomyces cerevisiae , that the protein is primarily involved in promoting mitosis by modulating the transcriptional activation of the APC/C protein–ubiquitin ligase activator Cdc20. Cks1 is required for both the periodic dissociation of Cdc28 kinase from the CDC20 promoter and the periodic association of the proteasome with the promoter. We propose that the essential role of Cks1 is to recruit the proteasome to, and/or dissociate the Cdc28 kinase from, the CDC20 promoter, thus facilitating transcription by remodelling transcriptional complexes or chromatin associated with the CDC20 gene.

Key Points Mammalian AMP-activated protein kinase (AMPK) was discovered as a protein kinase that switches off lipid synthesis. The yeast orthologue, the SNF1 complex, was discovered in screens for mutations that caused failure to grow on carbon sources other than glucose. The AMPK/SNF1 kinases exist as heterotrimeric complexes that are composed of catalytic α-subunits, β-subunits that bind to glycogen particles, and γ-subunits with tandem domains that bind AMP or ATP. Recent crystal structures are providing insights into the interactions between these domains and subunits. Binding of AMP activates the mammalian AMPK complex by causing allosteric activation and by inhibiting dephosphorylation of the critical activating site that is phosphorylated by upstream kinases. Upstream kinases that have recently been identified include the tumour suppressor LKB1 and calmodulin-dependent kinase kinase-β. Mutations in the γ2 isoforms that cause human heart disease interfere with the binding of the regulatory nucleotides AMP and ATP. AMP binding to wild-type AMPK appears to prevent the interaction of an inhibitory pseudosubstrate sequence (which is located in the N-terminal AMP-binding domain) with the substrate-binding groove. AMPK phosphorylates metabolic enzymes, transcription factors and co-activators that promote ATP-producing catabolic pathways and inhibit ATP-consuming biosynthetic pathways. It also inhibits cell growth and proliferation by triggering phosphorylation events that inhibit the TOR (target of rapamycin) pathway and cause stabilization of cell-cycle inhibitors such as p53 and p27. The extension of lifespan in response to sublethal stresses, such as caloric restriction, requires a specific isoform of AMPK in Caenorhabditis elegans . Recent unexpected findings also suggest that AMPK is involved in the establishment of cell polarity in insects as well as mammals. Maintaining the balance between ATP production and consumption is essential for cell survival. AMP-activated protein kinase (AMPK) is an energy sensor that, when energy levels are low, stimulates catabolism to produce ATP and inhibits biosynthesis and proliferation to conserve ATP. The SNF1/AMP-activated protein kinase (AMPK) family maintains the balance between ATP production and consumption in all eukaryotic cells. The kinases are heterotrimers that comprise a catalytic subunit and regulatory subunits that sense cellular energy levels. When energy status is compromised, the system activates catabolic pathways and switches off protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation. Surprisingly, recent results indicate that the AMPK system is also important in functions that go beyond the regulation of energy homeostasis, such as the maintenance of cell polarity in epithelial cells.

Two‐component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ c ). Increasing osmolality decreased deuterium exchange localized to the four‐helix bundle containing the autophosphorylation site (His 243 ). EnvZ c exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His 243 , suggesting that these two events are linked. In‐vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H‐bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli. The bacterial two‐component system EnvZ/OmpR governs the osmotic stress response. Increasing osmolality enhances intrahelical H‐bonding in the transmembrane kinase EnvZ, leading to helix stabilization, autophosphorylation and activation of downstream signalling.

Christopher Kirk and his colleagues have developed the first specific inhibitor of the immunoproteasome. They find that the immunoproteasome has a major role in regulating cytokine production, as well as antigen presentation, and their inhibitor has good efficacy in animal models of arthritis. The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low–molecular mass polypeptide-7 (LMP7, encoded by Psmb8 ), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I–restricted antigens in vitro and in vivo . Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-γ and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.

Key Points The proteasome is an abundant, catalytic complex that is found in both the nucleus and cytoplasm of eukaryotic cells. The function of the proteasome is to degrade or process intracellular proteins, some of which represent mediators of cell-cycle progression and apoptosis, such as the cyclins, caspases, BCL2 and nuclear factor of κB (NF-κB). Malignant cells are more susceptible to certain proteasome inhibitors, which might be explained, in part, by the reversal or bypass of some of the effects of the mutations in cell-cycle and apoptotic checkpoints that have led to tumorigenesis. Other explanations for this differential susceptibility include higher dependency of highly proliferative malignant cells on the proteasome system to remove aberrant proteins and the dependence of some tumours on the proteasome-dependent NF-κB activation pathway to maintain drug or radiation resistance. In addition to direct apoptotic effects, there is a strong biological basis for using proteasome inhibition to enhance sensitivity to standard chemotherapy and radiation therapy, and to overcome drug resistance. The proteasome inhibitor bortezomib has established clinical efficacy and an approved clinical indication for the treatment of relapsed and refractory multiple myeloma — proof of the principle that the proteasome is a suitable antineoplastic target. The proteasome is an abundant multi-enzyme complex that provides the main pathway for degradation of intracellular proteins in eukaryotic cells. As such, it controls the levels of proteins that are important for cell-cycle progression and apoptosis in normal and malignant cells; for example, cyclins, caspases, BCL2 and nuclear factor of κB. A proteasome inhibitor — bortezomib — has been developed that has shown efficacy as an anticancer agent in the clinic. How can targeting such a universal, broadly active cellular component provide the selectivity and specificity that are required for cancer therapeutics?

C-terminal α-amidation is the final and essential step in the biosynthesis of several peptide hormones. Peptidylglycine α-amidating monooxygenase (PAM) is the only known enzyme to catalyse this reaction. PAM amidating activity (AMA) is known to be present in human circulation, but its physiological role and significance as a clinical biomarker remains unclear. We developed a PAM-specific amidation assay that utilizes the naturally occurring substrate Adrenomedullin-Gly (ADM-Gly, 1–53). Using our amidation assay we quantified serum amidating activities in a large population-based cohort of more than 4900 individuals. A correlation of serum amidating activity with several clinical parameters including high blood pressure was observed. Increasing PAM-AMA was an independent predictor of hard outcomes related to hemodynamic stress such as cardiovascular mortality, atrial fibrillation and heart failure during long-term follow-up (8.8 ± 2.5 years). Moreover, results from an animal study in rats utilizing recombinant human PAM provide novel insights into the physiological role of circulating PAM and show its potential significance in circulating peptide amidation.

Epigenetic gene silencing is one of the fundamental mechanisms for ensuring proper gene expression patterns during cellular differentiation and development. Histone deacetylases (HDACs) are evolutionally conserved enzymes that remove acetyl modifications from histones and play a central role in epigenetic gene silencing. In cells, HDAC forms a multiprotein complex (HDAC complex) in which the associated proteins are believed to help HDAC carry out its cellular functions. Though each HDAC complex contains distinct components, the presence of isoforms for some of the components expands the variety of complexes and the diversity of their cellular roles. Recent studies have also revealed a functional link between HDAC complexes and specific histone demethylases. In this paper, we summarize the distinct and cooperative roles of four class I HDAC complexes, Sin3, NuRD, CoREST, and NCoR/SMRT, with respect to their component diversity and their relationship with specific histone demethylases.

Background and aimsInflammatory bowel disease (IBD), comprising Crohn´s disease and ulcerative colitis, is characterised by chronic relapsing inflammation of the gut. Increased proteasome activity, associated with the expression of immunoproteasomes, was found to enhance proinflammatory signalling and thus promotes inflammation in patients with IBD. The aim of this study was to explore whether modulation of the proteasomal activity is a suitable therapeutic approach to limit inflammation in colitis.MethodsThis concept was assessed in two different experimental set-ups. Development of dextran sodium sulfate (DSS)-induced colitis was tested (1) in lmp7−/− mice lacking the immunoproteasome subunit LMP7 and (2) in wild-type (WT) mice treated with the proteasome inhibitor bortezomib.ResultsCompared with WT mice, lmp7−/− mice develop significantly attenuated colitis due to reduced nuclear factor-κB (NF-κB) signalling in the absence of LMP7. Further, treatment with bortezomib revealed dose-dependent amelioration of DSS-induced inflammation. In both approaches modulation of the proteasome activity limited the secretion of proinflammatory cytokines and chemokines. Consequently, infiltration of the colon by neutrophils and expansion of inflammatory T helper 1 (Th1) and Th17 T cells was diminished and thus prevented excessive tissue damage.ConclusionsIt was demonstrated that modulation of the proteasome activity is effective in attenuating experimental colitis. The results reveal that reduction of the proteasome activity either by partial inhibition with bortezomib or by specifically targeting the immunoproteasome subunit LMP7 is a suitable treatment of intestinal inflammation.

Key Points The 26S proteasome is the most famous member of a family of 'chambered proteases' — multi-subunit enzymes that share a barrel-shaped structure and interior active sites that can only be accessed through a gated pore. The architecture of these enzymes promotes processive substrate degradation and makes substrate unfolding a prerequisite for proteolysis. Chambered proteases therefore have dedicated chaperone (regulatory) complexes that confer the ability to recognize and unfold cognate substrates. The substrates of each protease display a signal(s) that is recognized by its regulatory complex. Chambered proteases are exquisitely specific because the signals that they recognize are independent of the proteolytic cleavage sites Unlike prokaryotic and archaebacterial family members, which usually recognize primary sequence motifs in the substrate in a direct manner, the eukaryotic 26S proteasome typically recognizes substrates that are tagged with a 'polyubiquitin chain' — a polymer assembled from the small, conserved protein ubiquitin. Eukaryotes have a vast array of enzymes that mediate the highly regulated process of polyubiquitin tagging. Protease regulatory complexes carry out numerous functions, including recognition of substrate-based signals, unfolding of the substrate polypeptide chain, and gating of the protease pore. The regulatory (19S) complex of the 26S proteasome must also remove the polyubiquitin-chain signal, the constituent ubiquitins of which are then released and re-used. Recent studies of the protein unfolding that is catalysed by protease regulatory complexes indicate that signal recognition, unfolding and translocation are intimately coupled and intrinsically energy dependent. Substrates of prokaryotic regulatory complexes seem to start unfolding at the degradation signal; this is not necessarily the case for the 26S proteasome. In eukaryotes, the processes of signal (polyubiquitin) recognition and removal are surprisingly complex. Polyubiquitin removal must be precisely coordinated with downstream events in order to ensure that tagged substrates are, in fact, degraded. Understanding the precise mechanisms of substrate unfolding by protease regulatory complexes is a goal of many laboratories at present, as is elucidating the specific functions of the subunits of the 19S regulatory complex. The answers to these and other questions could open new avenues for the design of proteasome-directed therapeutics. 'Chambered proteases', including the eukaryotic 26S proteasome, use the energy of ATP to drive the unfolding and translocation of a polypeptide substrate into a chamber of sequestered proteolytic active sites. These proteases have diverse functions and are found in all three kingdoms of life. Understanding chambered proteases requires answers to two questions — how do these remarkable machines select the correct target proteins and how do they bring about the processive degradation of these molecules?

Biological signaling systems produce an output, such as the level of a phosphorylated protein, in response to defined input signals. The output level as a function of the input level is called the system's input-output relation. One may ask whether this input-output relation is sensitive to changes in the concentrations of the system's components, such as proteins and ATP. Because component concentrations often vary from cell to cell, it might be expected that the input-output relation will likewise vary. If this is the case, different cells exposed to the same input signal will display different outputs. Such variability can be deleterious in systems where survival depends on accurate match of output to input. Here we suggest a mechanism that can provide input-output robustness, that is, an input-output relation that does not depend on variations in the concentrations of any of the system's components. The mechanism is based on certain bacterial signaling systems. It explains how specific molecular details can work together to provide robustness. Moreover, it suggests an approach that can help identify a wide family of nonequilibrium mechanisms that potentially have robust input-output relations.