Substitution of Asp29 with Asn29 in the metallochaperone UreE of Streptococcus thermophilus DSM 20617T increases the urease activity and anticipates urea hydrolysis during milk fermentation.

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

The SlyD metallochaperone targets iron-sulfur biogenesis pathways and the TCA cycle.

IMPORTANCE: Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis/trans-isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori, measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.

Copper chaperone antioxidant 1: multiple roles and a potential therapeutic target.

Copper (Cu) was recently demonstrated to play a critical role in cellular physiological and biochemical processes, including energy production and maintenance, antioxidation and enzymatic activity, and signal transduction. Antioxidant 1 (ATOX1), a chaperone of Cu previously named human ATX1 homologue (HAH1), has been found to play an indispensable role in maintaining cellular Cu homeostasis, antioxidative stress, and transcriptional regulation. In the past decade, it has also been found to be involved in a variety of diseases, including numerous neurodegenerative diseases, cancers, and metabolic diseases. Recently, increasing evidence has revealed that ATOX1 is involved in the regulation of cell migration, proliferation, autophagy, DNA damage repair (DDR), and death, as well as in organism development and reproduction. This review summarizes recent advances in the research on the diverse physiological and cytological functions of ATOX1 and the underlying mechanisms of its action in human health and diseases. The potential of ATOX1 as a therapeutic target is also discussed. This review aims to pose unanswered questions related to ATOX1 biology and explore the potential use of ATOX1 as a therapeutic target.

Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C.

The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

Metallochaperone OsHIPP9 is involved in the retention of cadmium and copper in rice.

Metallochaperones are a unique class of proteins that play crucial roles in metal homoeostasis and detoxification. However, few metallochaperones have been functionally characterised in rice. Heterologous expression of Heavy metal-associated Isoprenylated Plant Protein 9 (OsHIPP9), a metallochaperone, altered yeast tolerance to cadmium (Cd) and copper (Cu). We investigated the physiological role of OsHIPP9 in rice. OsHIPP9 was primarily expressed in the root exodermis and xylem region of enlarged vascular bundles (EVB) at nodes. KO of OsHIPP9 increased the Cd concentrations of the upper nodes and panicle, but decreased Cd in expanded leaves. KO of OsHIPP9 decreased Cu uptake and accumulation in rice. Constitutive OX of OsHIPP9 increased Cd and Cu accumulation in aboveground tissues and brown rice. OsHIPP9 showed binding capacity for Cd and Cu. We propose that OsHIPP9 has dual metallochaperone roles, chelating Cd in the xylem region of EVB for Cd retention in the nodes and chelating Cu in rice roots to aid Cu uptake.

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

Memo1 binds reduced copper ions, interacts with copper chaperone Atox1, and protects against copper-mediated redox activity in vitro.

The protein mediator of ERBB2-driven cell motility 1 (Memo1) is connected to many signaling pathways that play key roles in cancer. Memo1 was recently postulated to bind copper (Cu) ions and thereby promote the generation of reactive oxygen species (ROS) in cancer cells. Since the concentration of Cu as well as ROS are increased in cancer cells, both can be toxic if not well regulated. Here, we investigated the Cu-binding capacity of Memo1 using an array of biophysical methods at reducing as well as oxidizing conditions in vitro. We find that Memo1 coordinates two reduced Cu (Cu(I)) ions per protein, and, by doing so, the metal ions are shielded from ROS generation. In support of biological relevance, we show that the cytoplasmic Cu chaperone Atox1, which delivers Cu(I) in the secretory pathway, can interact with and exchange Cu(I) with Memo1 in vitro and that the two proteins exhibit spatial proximity in breast cancer cells. Thus, Memo1 appears to act as a Cu(I) chelator (perhaps shuttling the metal ion to Atox1 and the secretory path) that protects cells from Cu-mediated toxicity, such as uncontrolled formation of ROS. This Memo1 functionality may be a safety mechanism to cope with the increased demand of Cu ions in cancer cells.

Periplasmic oxidized-protein repair during copper stress in E. coli: A focus on the metallochaperone CusF.

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveals that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows conservation of copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins.

Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

The role of nucleoside triphosphate hydrolase metallochaperones in making metalloenzymes.

Metalloenzymes catalyze a diverse set of challenging chemical reactions that are essential for life. These metalloenzymes rely on a wide range of metallocofactors, from single metal ions to complicated metallic clusters. Incorporation of metal ions and metallocofactors into apo-proteins often requires the assistance of proteins known as metallochaperones. Nucleoside triphosphate hydrolases (NTPases) are one important class of metallochaperones and are found widely distributed throughout the domains of life. These proteins use the binding and hydrolysis of nucleoside triphosphates, either adenosine triphosphate or guanosine triphosphate, to carry out highly specific and regulated roles in the process of metalloenzyme maturation. Here, we review recent literature on NTPase metallochaperones and describe the current mechanistic proposals and available structural data. By using representative examples from each type of NTPase, we also illustrate the challenges in studying these complicated systems. We highlight open questions in the field and suggest future directions. This minireview is part of a special collection of articles in memory of Professor Deborah Zamble, a leader in the field of nickel biochemistry.

A role for bioinorganic chemistry in the reactivation of mutant p53 in cancer.

Metal ion dysregulation has been implicated in a number of diseases from neurodegeneration to cancer. While defective metal ion transport mechanisms are known to cause specific diseases of genetic origin, the role of metal dysregulation in many diseases has yet to be elucidated due to the complicated function (both good and bad!) of metal ions in the body. A breakdown in metal ion speciation can manifest in several ways from increased reactive oxygen species (ROS) generation to an increase in protein misfolding and aggregation. In this review, we will discuss the role of Zn in the proper function of the p53 protein in cancer. The p53 protein plays a critical role in the prevention of genome mutations via initiation of apoptosis, DNA repair, cell cycle arrest, anti-angiogenesis, and senescence pathways to avoid propagation of damaged cells. p53 is the most frequently mutated protein in cancer and almost all cancers exhibit malfunction along the p53 pathway. Thus, there has been considerable effort dedicated to restoring normal p53 expression and activity to mutant p53. This includes understanding the relative populations of the Zn-bound and Zn-free p53 in wild-type and mutant forms, and the development of metallochaperones to re-populate the Zn binding site to restore mutant p53 activity. Parallels will be made to the development of multifunctional metal binding agents for modulating the aggregation of the amyloid-beta peptide in Alzheimer's Disease (AD).

Metallochaperones: A critical regulator of metal homeostasis and beyond.

Metallochaperones are a class of unique protein families that was originally found to interact with cellular metal ions by metal delivery to specific target proteins such as metal enzymes. Recently, some members of metallochaperones receive much attention owning to their multi-biological functions in mediating plant growth, development and biotic or abiotic stress responses, particularly in the aspects of metal transport and accumulation in plants. For example, some non-essential toxic heavy metals (e.g. cadmium and mercury) accumulating in farmland due to the industrial and agronomic activities, are a constant threat to crop production, food safety and human health. Digging genetic resources and functional genes like metallochaperones is critical for understanding the metal detoxification in plants, and may help develop cleaner crops with minimal toxic metals in leafy vegetables and grains, or plants for metal-polluted soil phytoremediation. In this review, we highlight the current advancement of the research on functions of metallochaperones in metal accumulation, detoxification and homeostasis. We also summarize the recent progress of the research on the critical roles of the metal-binding proteins in regulating plant responses to some other biological processes including plant growth, development, pathogen stresses, and abiotic stresses such salt, drought, cold and light. Finally, an additional capacity of some members of metallochaperones involved in the resistance to the pathogen attack and possibly regulatory roles was reviewed.

Revisiting the CooJ family, a potential chaperone for nickel delivery to [NiFe]­carbon monoxide dehydrogenase.

Nickel insertion into nickel-dependent carbon monoxide dehydrogenase (CODH) represents a key step in the enzyme activation. This is the last step of the biosynthesis of the active site, which contains an atypical heteronuclear NiFe4S4 cluster known as the C-cluster. The enzyme maturation is performed by three accessory proteins, namely CooC, CooT and CooJ. Among them, CooJ from Rhodospirillum rubrum is a histidine-rich protein containing two distinct and spatially separated Ni(II)-binding sites: a N-terminal high affinity site (HAS) and a histidine tail at the C-terminus. In 46 CooJ homologues, the HAS motif was found to be strictly conserved with a H(W/F)XXHXXXH sequence. Here, a proteome database search identified at least 150 CooJ homologues and revealed distinct motifs for HAS, featuring 2, 3 or 4 histidines. The purification and biophysical characterization of three representative members of this protein family showed that they are all homodimers able to bind Ni(II) ions via one or two independent binding sites. Initially thought to be present only in R. rubrum, this study strongly suggests that CooJ could play a significant role in CODH maturation or in nickel homeostasis.

COG0523 proteins: a functionally diverse family of transition metal-regulated G3E P-loop GTP hydrolases from bacteria to man.

Transition metal homeostasis ensures that cells and organisms obtain sufficient metal to meet cellular demand while dispensing with any excess so as to avoid toxicity. In bacteria, zinc restriction induces the expression of one or more Zur (zinc-uptake repressor)-regulated Cluster of Orthologous Groups (COG) COG0523 proteins. COG0523 proteins encompass a poorly understood sub-family of G3E P-loop small GTPases, others of which are known to function as metallochaperones in the maturation of cobalamin (CoII) and NiII cofactor-containing metalloenzymes. Here, we use genomic enzymology tools to functionally analyse over 80 000 sequences that are evolutionarily related to Acinetobacter baumannii ZigA (Zur-inducible GTPase), a COG0523 protein and candidate zinc metallochaperone. These sequences segregate into distinct sequence similarity network (SSN) clusters, exemplified by the ZnII-Zur-regulated and FeIII-nitrile hydratase activator CxCC (C, Cys; X, any amino acid)-containing COG0523 proteins (SSN cluster 1), NiII-UreG (clusters 2, 8), CoII-CobW (cluster 4), and NiII-HypB (cluster 5). A total of five large clusters that comprise ≈ 25% of all sequences, including cluster 3 which harbors the only structurally characterized COG0523 protein, Escherichia coli YjiA, and many uncharacterized eukaryotic COG0523 proteins. We also establish that mycobacterial-specific protein Y (Mpy) recruitment factor (Mrf), which promotes ribosome hibernation in actinomycetes under conditions of ZnII starvation, segregates into a fifth SSN cluster (cluster 17). Mrf is a COG0523 paralog that lacks all GTP-binding determinants as well as the ZnII-coordinating Cys found in CxCC-containing COG0523 proteins. On the basis of this analysis, we discuss new perspectives on the COG0523 proteins as cellular reporters of widespread nutrient stress induced by ZnII limitation.

The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity.

Tyrosinase (Ty) and catechol oxidase (CO) are members of type-3 copper enzymes. While Ty catalyzes both phenolase and catecholase reactions, CO catalyzes only the latter reaction. In the present study, Ty was found to catalyze the catecholase reaction, but hardly the phenolase reaction in the presence of the metallochaperon called "caddie protein (Cad)". The ability of the substrates to dissociate the motif shielding the active-site pocket seems to contribute critically to the substrate specificity of Ty. In addition, a mutation at the N191 residue, which forms a hydrogen bond with a water molecule near the active center, decreased the inherent ratio of phenolase versus catecholase activity. Unlike the wild-type complex, reaction intermediates were not observed when the catalytic reaction toward the Y98 residue of Cad was progressed in the crystalline state. The increased basicity of the water molecule may be necessary to inhibit the proton transfer from the conjugate acid to a hydroxide ion bridging the two copper ions. The deprotonation of the substrate hydroxyl by the bridging hydroxide seems to be significant for the efficient catalytic cycle of the phenolase reaction.

A novel mode of control of nickel uptake by a multifunctional metallochaperone.

Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.

Iron Chaperone Poly rC Binding Protein 1 Protects Mouse Liver From Lipid Peroxidation and Steatosis.

BACKGROUND AND AIMS: Iron is essential yet also highly chemically reactive and potentially toxic. The mechanisms that allow cells to use iron safely are not clear; defects in iron management are a causative factor in the cell-death pathway known as ferroptosis. Poly rC binding protein 1 (PCBP1) is a multifunctional protein that serves as a cytosolic iron chaperone, binding and transferring iron to recipient proteins in mammalian cells. Although PCBP1 distributes iron in cells, its role in managing iron in mammalian tissues remains open for study. The liver is highly specialized for iron uptake, utilization, storage, and secretion. APPROACH AND RESULTS: Mice lacking PCBP1 in hepatocytes exhibited defects in liver iron homeostasis with low levels of liver iron, reduced activity of iron enzymes, and misregulation of the cell-autonomous iron regulatory system. These mice spontaneously developed liver disease with hepatic steatosis, inflammation, and degeneration. Transcriptome analysis indicated activation of lipid biosynthetic and oxidative-stress response pathways, including the antiferroptotic mediator, glutathione peroxidase type 4. Although PCBP1-deleted livers were iron deficient, dietary iron supplementation did not prevent steatosis; instead, dietary iron restriction and antioxidant therapy with vitamin E prevented liver disease. PCBP1-deleted hepatocytes exhibited increased labile iron and production of reactive oxygen species (ROS), were hypersensitive to iron and pro-oxidants, and accumulated oxidatively damaged lipids because of the reactivity of unchaperoned iron. CONCLUSIONS: Unchaperoned iron in PCBP1-deleted mouse hepatocytes leads to production of ROS, resulting in lipid peroxidation (LPO) and steatosis in the absence of iron overload. The iron chaperone activity of PCBP1 is therefore critical for limiting the toxicity of cytosolic iron and may be a key factor in preventing the LPO that triggers the ferroptotic cell-death pathway.

Phytochrome B and PCH1 protein dynamics store night temperature information.

Plants experience temperature fluctuations during the course of the daily cycle, and although stem growth responds rapidly to these changes we largely ignore whether there is a short-term memory of previous conditions. Here we show that nighttime temperatures affect the growth of the hypocotyl of Arabidopsis thaliana seedlings not only during the night but also during the subsequent photoperiod. Active phytochrome B (phyB) represses nighttime growth and warm temperatures reduce active phyB via thermal reversion. The function of PHOTOPERIODIC CONTROL OF HYPOCOTYL1 (PCH1) is to stabilise active phyB in nuclear bodies but, surprisingly, warmth reduces PCH1 gene expression and PCH1 stability. When phyB was active at the beginning of the night, warm night temperatures enhanced the levels of nuclear phyB and reduced hypocotyl growth rate during the following day. However, when end-of-day far-red light minimised phyB activity, warm night temperatures reduced the levels of nuclear phyB and enhanced the hypocotyl growth rate during the following day. This complex growth pattern was absent in the phyB mutant. We propose that temperature-induced changes in the levels of PCH1 and in the size of the physiologically relevant nuclear pool of phyB amplify the impact of phyB-mediated temperature sensing.

Structural insights into Fe-S protein biogenesis by the CIA targeting complex.

The cytosolic iron-sulfur (Fe-S) assembly (CIA) pathway is required for the insertion of Fe-S clusters into cytosolic and nuclear client proteins, including many DNA replication and repair factors. The molecular mechanisms of client protein recognition and Fe-S cluster transfer remain unknown. Here, we report crystal structures of the CIA targeting complex (CTC), revealing that its CIAO2B subunit is centrally located and bridges CIAO1 and the client adaptor protein MMS19. Cryo-EM reconstructions of human CTC bound either to the DNA replication factor primase or to the DNA helicase DNA2, combined with biochemical, biophysical and yeast complementation assays, reveal an evolutionarily conserved, bipartite client recognition mode facilitated by CIAO1 and the structural flexibility of the MMS19 subunit. Unexpectedly, the primase Fe-S cluster is located ~70 Å away from the CTC reactive cysteine, implicating conformational dynamics of the CTC or additional maturation factors in the mechanism of Fe-S cluster transfer.

Ciao1 interacts with Crumbs and Xpd to regulate organ growth in Drosophila.

Ciao1 is a component of the cytosolic iron-sulfur cluster assembly (CIA) complex along with MMS19 and MIP18. Xeroderma pigmentosum group D (XPD), a DNA helicase involved in regulation of cell cycle and transcription, is a CIA target for iron-sulfur (Fe/S) modification. In vivo function of Ciao1 and Xpd in developing animals has been rarely studied. Here, we reveal that Ciao1 interacts with Crumbs (Crb), Galla, and Xpd to regulate organ growth in Drosophila. Abnormal growth of eye by overexpressing Crb intracellular domain (Crbintra) is suppressed by reducing the Ciao1 level. Loss of Ciao1 or Xpd causes similar impairment in organ growth. RNAi knockdown of both Ciao1 and Xpd show similar phenotypes as Ciao1 or Xpd RNAi alone, suggesting their function in a pathway. Growth defects caused by Ciao1 RNAi are suppressed by overexpression of Xpd. Ciao1 physically interacts with Crbintra, Galla, and Xpd, supporting their genetic interactions. Remarkably, Xpd RNAi defects can also be suppressed by Ciao1 overexpression, implying a mutual regulation between the two genes. Ciao1 mutant clones in imaginal discs show decreased levels of Cyclin E (CycE) and death-associated inhibitor of apoptosis 1 (Diap1). Xpd mutant clones share the similar reduction of CycE and Diap1. Consequently, knockdown of Ciao1 and Xpd by RNAi show increased apoptotic cell death. Further, CycE overexpression is sufficient to restore the growth defects from Ciao1 RNAi or Xpd RNAi. Interestingly, Diap1 overexpression in Ciao1 mutant clones induces CycE expression, suggesting that reduced CycE in Ciao1 mutant cells is secondary to loss of Diap1. Taken together, this study reveals new roles of Ciao1 and Xpd in cell survival and growth through regulating Diap1 level during organ development.