Julia I. Toth

REDD1, an inhibitor of mTOR signalling, is regulated by the CUL4A–DDB1 ubiquitin ligase

The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia‐inducible factor‐1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post‐translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin‐mediated degradation mediated by the CUL4A–DDB1–ROC1–β‐TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3β. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.

Selective Coactivator Interactions in Gene Activation by SREBP-1a and -1c

ABSTRACT Requisite levels of intracellular cholesterol and fatty acids are maintained in part by the sterol regulatory element binding proteins (SREBPs). Three major SREBP isoforms exist; SREBP-1a and SREBP-1c are expressed from overlapping mRNAs, whereas SREBP-2 is encoded by a separate gene. The active forms of SREBP-1a and SREBP-1c differ only at their extreme N termini; SREBP-1c lacks 28 aa present in SREBP-1a and instead contains 4 unique aa of its own. While the SREBP-1a and -1c isoforms differentially activate transcription, the molecular basis of this difference is unknown. Here we define the differences between these proteins that confer the enhanced activity of SREBP-1a and demonstrate that this enhancement is a direct result of its avid binding to the coactivator CREB binding protein (CBP) and the mammalian mediator complex. While previous work determined that the C/H1 zinc finger and KIX domains of CBP bind to SREBP-1a, we provide evidence that the interaction with C/H1 is important for gene activation. We further show that the association between the activation domain of SREBP-1 and mediator is through aa 500 to 824 of DRIP150. Finally, we demonstrate the recruitment of mediator to an SREBP-responsive promoter in a sterol-dependent manner.

CAND1 controls in vivo dynamics of the cullin 1-RING ubiquitin ligase repertoire

The combinatorial architecture of cullin 1-RING ubiquitin ligases (CRL1s), in which multiple F-box containing substrate receptors (FBPs) compete for access to CUL1, poses special challenges to assembling CRL1 complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire FBP repertoire. Here, using highly quantitative mass spectrometry, we demonstrate that this problem is addressed by CAND1, a factor that controls the dynamics of the global CRL1 network by promoting the assembly of newly synthesized FBPs with CUL1-RBX1 core complexes. Our studies of in vivo CRL1 dynamics and in vitro biochemical findings showing that CAND1 can displace FBPs from Cul1p suggest that CAND1 functions in a cycle that serves to exchange FBPs on CUL1 cores. We propose that this cycle assures comprehensive sampling of the entire FBP repertoire in order to maintain the CRL1 landscape, a function that we show to be critical for substrate degradation and normal physiology.

Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement

The classical and lectin pathways of complement activation neutralize pathogens and stimulate key immunological processes. Both pathways are initiated by collagen-containing, soluble pattern recognition molecules associated with specific serine proteases. In the classical pathway, C1q binds to Ab-Ag complexes or bacterial surfaces to activate C1r and C1s. In the lectin pathway, mannan-binding lectin and ficolins bind to carbohydrates on pathogens to activate mannan-binding lectin-associated serine protease 2. To characterize the interactions leading to classical pathway activation, we have analyzed binding between human C1q, C1r, and C1s, which associate to form C1, using full-length and truncated protease components. We show that C1r and C1s bind to C1q independently. The CUB1-epidermal growth factor fragments contribute most toward binding, but CUB2 of C1r, but not of C1s, is also important. Each C1rs tetramer presents a total of six binding sites, one for each of the collagenous domains of C1q. We also demonstrate that subcomponents of the lectin and classical pathways cross-interact. Thus, although the stoichiometries of complexes differ, interactions are analogous, with equivalent contacts between recognition and protease subcomponents. Importantly, these new data are contrary to existing models of C1 and enable us to propose a new model using mannan-binding lectin-mannan-binding lectin-associated serine protease interactions as a template.

A Gatekeeper Residue for NEDD8-Activating Enzyme Inhibition by MLN4924

Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzymes affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecules inhibitory effects, allowing cancer cell survival.

Selective Association of Sterol Regulatory Element-binding Protein Isoforms with Target Promoters in Vivo

The mRNAs for all three members of the sterol regulatory element-binding protein (SREBP) family are widely expressed, and the proteins are highly similar. They have potential to both hetero- and homodimerize through their bHLHLZ domains, so it has been difficult to definitively study the role of each one apart from the other two. In the current study, we have utilized cell lines that express only one functional SREBP and the chromatin immunoprecipitation technique to analyze individual SREBP binding to three specific target genes: hydroxymethylglutaryl-CoA reductase (Red), fatty acid synthase (FAS), and squalene synthase (SQS). Our studies show that SREBP-2 binds to promoters for all three genes, and in agreement with the original report using these cells, all three mRNAs are also induced. In the line expressing only SREBP-1a, mRNAs for Red and FAS are induced, but SQS is not. Chromatin immunoprecipitation also shows that SREBP-1a is recruited efficiently to Red and FAS promoters but not to SQS. This observation indicates SREBP-2 selectively binds the SQS promoter and is sufficient to explain the lack of SQS mRNA induction in the SREBP-1a-expressing cells. SREBP-1c protein was not stably recruited to any SREBP target promoter despite being fully active in DNA binding when purified from extracts of the corresponding cells. This is also sufficient to explain the lack of SREBP target gene induction by the singular expression of SREBP-1c. We also show that whereas SREBP-1a and -2 proteins interact efficiently with transcriptional co-activators that modify cellular chromatin, SREBP-1c does not. Taken together, our data support a model suggesting that chromatin modification is required during the initial stage of specific site recognition by SREBPs in native chromatin in vivo.

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine A possible means to regulate gene expression

Summary Chromosomal rearrangements apparently account for the presence of a primate‐specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72‐residue leader peptide on Isoform A, and a CUG site that codes for a 28‐residue leader peptide on Isoform B. We report studies that provide evidence for the N‐terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28‐amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.

Bifunctional Apoptosis Regulator (BAR), an Endoplasmic Reticulum (ER)-associated E3 Ubiquitin Ligase, Modulates BI-1 Protein Stability and Function in ER Stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.

Regional Distribution of Human Trypsinogen 4 in Human Brain at mRNA and Protein Level

Gene PRSS3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. Mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mRNA for trypsinogen 4 has recently been identified in brain and other human tissues. We measured the amount of trypsinogen 4 mRNA and the quantity of the protein as well in 17 selected areas of the human brain. Our data suggest that human trypsinogen 4 is widely but unevenly distributed in the human brain. By immunohistochemistry, here we show that this protease is localized in neurons and glial cells, predominantly in astrocytes. In addition to cellular immunoreactivity, human trypsinogen 4 immunopositive dots were detected in the extracellular matrix, supporting the view that human trypsinogen 4 might be released from the cells under special conditions.

Regulation of Endoplasmic Reticulum-associated Degradation by RNF5-dependent Ubiquitination of JNK-associated Membrane Protein (JAMP)

Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTRΔ508 and T cell receptor α, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.