Stephen Baird

The inhibitors of apoptosis (IAPs) and their emerging role in cancer.

The inhibitor of apoptosis protein family has been characterized over the past 5 years, initially in baculovirus and more recently in metazoans. The IAPs are a widely expressed gene family of apoptotic inhibitors from both phylogenic and physiologic points of view. The diversity of triggers against which the IAPs suppress apoptosis is greater than that observed for any other family of apoptotic inhibitors including the bcl-2 family. The central mechanisms of IAP apoptotic suppression appear to be through direct caspase and pro-caspase inhibition (primarily caspase 3 and 7) and modulation of and by the transcription factor NF-κB. Although evidence for a direct oncogenic role for the IAPs has yet to be delineated, a number of lines of evidence point towards this class of protein playing a role in oncogenesis. The strongest evidence for IAP involvement in cancer is seen in the IAP called survivin. Although not observed in adult differentiated tissue, survivin is present in most transformed cell lines and cancers tested to date. Survivin has been shown to inhibit caspase directly and apoptosis in general, moreover survivin protein levels correlate inversely with 5 year survival rates in colorectal cancer. Recent data has also implicated survivin in cell cycle control. The second line of evidence for IAP involvement in cancer comes from their emerging role as mediators and regulators of the anti-apoptotic activity of v-Rel and NF-κB transcription factor families. The IAPs have been shown to be induced by NF-κB or v-Rel in multiple cell lines and conversely, HIAP1 and HIAP2 have been shown to activate NF-κB possibly forming a positive feed-back loop. Overall a picture consistent with an IAP role in tumour progression rather than tumour initiation is emerging making the IAPs an attractive therapeutic target.

The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

IAP-targeted therapies for cancer.

DNA damage, chromosomal abnormalities, oncogene activation, viral infection, substrate detachment and hypoxia can all trigger apoptosis in normal cells. However, cancer cells acquire mutations that allow them to survive these threats that are part and parcel of the transformation process or that may affect the growth and dissemination of the tumor. Eventually, cancer cells accumulate further mutations that make them resistant to apoptosis mediated by standard cytotoxic chemotherapy or radiotherapy. The inhibitor of apoptosis (IAP) family members, defined by the presence of a baculovirus IAP repeat (BIR) protein domain, are key regulators of cytokinesis, apoptosis and signal transduction. Specific IAPs regulate either cell division, caspase activity or survival pathways mediated through binding to their BIR domains, and/or through their ubiquitin-ligase RING domain activity. These protein–protein interactions and post-translational modifications are the subject of intense investigations that shed light on how these proteins contribute to oncogenesis and resistance to therapy. In the past several years, we have seen multiple approaches of IAP antagonism enter the clinic, and the rewards of such strategies are about to reap benefit. Significantly, small molecule pan-IAP antagonists that mimic an endogenous inhibitor of the IAPs, called Smac, have demonstrated an unexpected ability to sensitize cancer cells to tumor necrosis factor-α and to promote autocrine or paracrine production of this cytokine by the tumor cell and possibly, other cells too. This review will focus on these and other developmental therapeutics that target the IAPs in cancer.

A search for structurally similar cellular internal ribosome entry sites

Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function.

ROMO1 Is an Essential Redox-Dependent Regulator of Mitochondrial Dynamics

ROMO1 links the oxidative state of the cell to changes in mitochondrial shape and function. Fueling Fusion Mitochondria are dynamic organelles that undergo fusion or fission. In response to cell death–inducing stimuli, mitochondria undergo fragmentation. OPA1 is a guanosine triphosphatase (GTPase) that is present as a transmembrane protein in the inner mitochondrial membrane and as a cleaved form in the intermembrane space; a balance in the abundance of both forms is required for OPA1 to promote mitochondrial fusion. Norton et al. identified ROMO1 as a regulator of mitochondrial morphology that, in response to reactive oxygen species, was oxidized and formed inactive oligomers. Cells lacking ROMO1 had more of the cleaved form of OPA1, showed an increase in fragmented mitochondria, and were more sensitive to cell death–inducing stimuli. Thus, ROMO1 acts as a link between the oxidative state of the cell and the changes in mitochondrial shape and function. The dynamics of mitochondria undergoing fusion and fragmentation govern many mitochondrial functions, including the regulation of cell survival. Although the machinery that catalyzes fusion and fragmentation has been well described, less is known about the signaling components that regulate these phenomena. We performed a genome-wide RNA interference (RNAi) screen and identified reactive oxygen species modulator 1 (ROMO1) as a redox-regulated protein required for mitochondrial fusion and normal cristae morphology. We showed that oxidative stress promoted the formation of high–molecular weight ROMO1 complexes and that knockdown of ROMO1 promoted mitochondrial fission. ROMO1 was essential for the oligomerization of the inner membrane guanosine triphosphatase (GTPase) OPA1, which is required to maintain the integrity of cristae junctions. As a consequence, cells lacking ROMO1 displayed fragmented mitochondria and loss of cristae, causing impaired mitochondrial respiration and increased sensitivity to cell death stimuli. Together, our data identify ROMO1 as a critical molecular switch that couples metabolic stress and mitochondrial morphology, linking mitochondrial fusion to cell survival.

NF45 functions as an IRES trans-acting factor that is required for translation of cIAP1 during the unfolded protein response.

Expression of the cellular inhibitor of apoptosis protein 1 (cIAP1) is unexpectedly repressed at the level of translation under normal physiological conditions in many cell lines. We have previously shown that the 5′ untranslated region of cIAP1 mRNA contains a stress-inducible internal ribosome entry site (IRES) that governs expression of cIAP1 protein. Although inactive in unstressed cells, the IRES supports cap-independent translation of cIAP1 in response to endoplasmic reticulum stress. To gain an insight into the mechanism of cIAP1 IRES function, we empirically derived the minimal free energy secondary structure of the cIAP1 IRES using enzymatic cleavage mapping. We subsequently used RNA affinity chromatography to identify several cellular proteins, including nuclear factor 45 (NF45) as cIAP1 IRES binding proteins. In this report we show that NF45 is a novel RNA binding protein that enhances IRES-dependent translation of endogenous cIAP1. Further, we show that NF45 is required for IRES-mediated induction of cIAP1 protein during the unfolded protein response. The data presented are consistent with a model in which translation of cIAP1 is governed, at least in part, by NF45, a novel cellular IRES trans-acting factor.

Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy

Mitochondrial dysfunction is a hallmark of aging and numerous human diseases, including Parkinson disease (PD). Multiple homeostatic mechanisms exist to ensure mitochondrial integrity, including the selective autophagic program mitophagy, that is activated during starvation or in response to mitochondrial dysfunction. Following prolonged loss of potential across the inner mitochondrial membrane (ΔΨ), PTEN-induced putative kinase 1 (PINK1) and the E3-ubiquitin ligase PARK2 work in the same pathway to trigger mitophagy of dysfunctional mitochondria. Mutations in PINK1 and PARK2, as well as PARK7/DJ-1, underlie autosomal recessive Parkinsonism and impair mitochondrial function and morphology. In a genome-wide RNAi screen searching for genes that are required for PARK2 translocation to the mitochondria, we identified ATPase inhibitory factor 1 (ATPIF1/IF1) as essential for PARK2 recruitment and mitophagy in cultured cells. During uncoupling, ATPIF1 promotes collapse of ΔΨ and activation of the PINK-PARK2 mitophagy pathway by blocking the ATPase activity of the F1-Fo ATP synthase. Restoration of ATPIF1 in Rho0 cells, which lack mtDNA and a functional electron transport chain, lowers ΔΨ and triggers PARK2 recruitment. Our findings identified ATPIF1 and the ATP synthase as novel components of the PINK1-PARK2 mitophagy pathway and provide genetic evidence that loss of ΔΨ is an essential trigger for mitophagy.

DNM1L-related mitochondrial fission defect presenting as refractory epilepsy.

Mitochondrial fission and fusion are dynamic processes vital to mitochondrial quality control and the maintenance of cellular respiration. In dividing mitochondria, membrane scission is accomplished by a dynamin-related GTPase, DNM1L, that oligomerizes at the site of fission and constricts in a GTP-dependent manner. There is only a single previous report of DNM1L-related clinical disease: a female neonate with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF; OMIM #614388), a lethal disorder characterized by cerebral dysgenesis, seizures, lactic acidosis, elevated very long chain fatty acids, and abnormally elongated mitochondria and peroxisomes. Here, we describe a second individual, diagnosed via whole-exome sequencing, who presented with developmental delay, refractory epilepsy, prolonged survival, and no evidence of mitochondrial or peroxisomal dysfunction on standard screening investigations in blood and urine. EEG was nonspecific, showing background slowing with frequent epileptiform activity at the frontal and central head regions. Electron microscopy of skeletal muscle showed subtle, nonspecific abnormalities of cristal organization, and confocal microscopy of patient fibroblasts showed striking hyperfusion of the mitochondrial network. A panel of further bioenergetic studies in patient fibroblasts showed no significant differences versus controls. The proband’s de novo DNM1L variant, NM_012062.4:c.1085G>A; NP_036192.2:p.(Gly362Asp), falls within the middle (oligomerization) domain of DNM1L, implying a likely dominant-negative mechanism. This disorder, which presents nonspecifically and affords few diagnostic clues, can be diagnosed by means of DNM1L sequencing and/or confocal microscopy.

Nucleotide composition of cellular internal ribosome entry sites defines dependence on NF45 and predicts a posttranscriptional mitotic regulon.

ABSTRACT The vast majority of cellular mRNAs initiate their translations through a well-defined mechanism of ribosome recruitment that occurs at the 5′-terminal 7-methylguanosine cap with the help of several canonical protein factors. A subset of cellular and viral mRNAs contain regulatory motifs in their 5′ untranslated regions (UTRs), termed internal ribosome entry sites (IRES), that sidestep this canonical mode of initiation. On cellular mRNAs, this mechanism requires IRES trans-acting protein factors (ITAFs) that facilitate ribosome recruitment downstream of the cap. While several ITAFs and their target mRNAs have been empirically identified, the in silico prediction of targets has proved difficult. Here, we report that a high AU content (>60%) of the IRES-containing 5′ UTRs serves as an excellent predictor of dependence on NF45, a recently identified ITAF. Moreover, we provide evidence that cells deficient in NF45 ITAF activity exhibit reduced IRES-mediated translation of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 (cIAP1) mRNAs that, in turn, leads to dysregulated expression of their respective targets, survivin and cyclin E. This specific defect in IRES translation explains in part the cytokinesis impairment and senescence-like phenotype observed in HeLa cells expressing NF45 RNA interference (RNAi). This study uncovers a novel role for NF45 in regulating ploidy and highlights the importance of IRES-mediated translation in cellular homeostasis.

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation.

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.