Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development.

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.

RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.

N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity.

The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier.

Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.

Impact of Mitochondrial Fatty Acid Synthesis on Mitochondrial Biogenesis.

Biology students today are taught that mitochondria are 'the powerhouse of the cell'. This gross over-simplification of their cellular role has arguably led to a paucity of knowledge concerning the many other tasks carried out by this multifunctional organelle. Mitochondrial fatty acid synthesis (mtFAS) is one such under-appreciated pathway that is crucial for mitochondrial function, although even mitochondrial experts are often surprised to learn of its existence. For many years, the only function of mtFAS was thought to be the production of lipoic acid, an important co-factor for several mitochondrial enzymes. However, recent advances have revealed a far wider role for mtFAS in mitochondrial physiology. The discovery of human patients with mutations in mtFAS enzymes has brought renewed interest in understanding the full significance of this novel mode of mitochondrial metabolic regulation. We now appreciate that mtFAS is a nutrient-sensitive pathway that provides an elegant mechanism whereby acetyl-CoA regulates its own consumption via coordination of lipoic acid synthesis and tricarboxylic acid (TCA) cycle activity, iron-sulfur (FeS) cluster biogenesis, assembly of oxidative phosphorylation complexes, and mitochondrial translation. In this minireview, we describe and build upon the important discoveries that led to our current understanding of this elegant mechanism of coordination of nutrient status and metabolism.

The Force Is Strong with This One: Metabolism (Over)powers Stem Cell Fate.

Compared to their differentiated progeny, stem cells are often characterized by distinct metabolic landscapes that emphasize anaerobic glycolysis and a lower fraction of mitochondrial carbohydrate oxidation. Until recently, the metabolic program of stem cells had been thought to be a byproduct of the environment, rather than an intrinsic feature determined by the cell itself. However, new studies highlight the impact of metabolic behavior on the maintenance and function of intestinal stem cells and hair follicle stem cells. This Review summarizes and discusses the evidence that metabolism is not a mere consequence of, but rather influential on stem cell fate.

RING-Between-RING E3 Ligases: Emerging Themes amid the Variations.

Covalent, reversible, post-translational modification of cellular proteins with the small modifier, ubiquitin (Ub), regulates virtually every known cellular process in eukaryotes. The process is carried out by a trio of enzymes: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme, and a Ub ligase (E3) enzyme. RING-in-Between-RING (RBR) E3s constitute one of three classes of E3 ligases and are defined by a RING-HECT-hybrid mechanism that utilizes a E2-binding RING domain and a second domain (called RING2) that contains an active site Cys required for the formation of an obligatory E3~Ub intermediate. Albeit a small class, RBR E3s in humans regulate diverse cellular process. This review focuses on non-Parkin members such as HOIP/HOIL-1L (the only E3s known to generate linear Ub chains), HHARI and TRIAD1, both of which have been recently demonstrated to work together with Cullin RING E3 ligases. We provide a brief historical background and highlight, summarize, and discuss recent developments in the young field of RBR E3s. Insights reviewed here include new understandings of the RBR Ub-transfer mechanism, specifically the role of RING1 and various Ub-binding sites, brief structural comparisons among members, and different modes of auto-inhibition and activation.

Structural Studies of HHARI/UbcH7∼Ub Reveal Unique E2∼Ub Conformational Restriction by RBR RING1.

RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2∼ubiquitin (E2∼Ub) via their RINGs but canonical RING E3s promote closed E2∼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2∼Ub to favor Ub transfer to the E3 active site. This different RING/E2∼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2∼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence.

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Structural Biology: Parkin's Serpentine Shape Revealed in the Year of the Snake.

Parkin is an E3 ubiquitin ligase, mutations in which are responsible for autosomal recessive juvenile parkinsonism. Recently, several structures of Parkin have been solved, revealing its serpentine shape and modes of auto-inhibition.