Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

Sub-families of related zinc finger protein genes have been defined on the basis of evolutionary conserved structural features found outside the C₂–H₂ finger repeats. Such elements include the FAX domain found in a large number of Xenopus ZFPs, the evolutionary conserved KRAB (Kriippel—associated box) and the ZiN (zinc finger N—terminal) domains. Here we describe a new evolutionarily conserved motif within zinc finger proteins which we have named the leucine rich region (LeR). Since conserved modules in regulatory proteins may specify properties relevant to their action we have determined the functional capabilities of LeR and the KRAB domains in the regulation of gene transcription by fusing relevant regions to a heterologous DNAbinding domain (GAL4 DNA—binding domain). We found that the KRAB—A domain tethered to RNA polymerase II promoters by a GAL4 DNA—binding domain actively represses transcription in a distance—independent manner. KRAB—mediated repression is dependent on the dose of the GAL4 — KRAB—A fusion protein and on the presence of GAL4 binding sites on the DNA. Conversely, the LeR domain did not modulate significantly the transcription. Our results indicate that the KRAB domain present in the non-finger region of many ZFP genes quenches transcription possibly due to specific protein—protein interactions between the KRAB-A domain and components of the proximal transcriptional apparatus.

Tight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the ΔNp63α protein. We found that MDM2 binds ΔNp63α in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for ΔNp63α nuclear export and subsequent degradation, whereas the MDM2 ring-finger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of ΔNp63α by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the α and β tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous ΔNp63α in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative ΔNp63α protein.

Genetic alteration of the p53 tumor suppressor gene, which monitors DNA damage and operates cell cycle checkpoints, is a major factor in the development of human malignancies. The p53 protein belongs to a family that also includes two structurally related proteins, p63 and p73. Although all three proteins share similar transcriptional functions and antiproliferative effects, each of them appears to play a distinct role in development and tumor suppression. One of the principal regulators of p53 activity is the MDM2 protein. The interaction of MDM2 with p53 inhibits p53 transcriptional activity and targets p53 for ubiquitin-dependent degradation. The ability of MDM2 to inhibit p53 functions is antagonized by the ARF oncosuppressor protein. We show here that like p53, the p63α and p63γ isoforms are able to associate with human MDM2 (HDM2). Overexpression of HDM2 increased the steady-state level of intracellular p63 and enhanced its transcriptional activity. Both effects appeared to be counteracted by ARF coexpression. These data indicate that p63 can be activated by HDM2 under conditions in which p53 is inhibited. Therefore, HDM2 expression could support p63-specific transcriptional functions on a common set of genes, keeping interference by p53 at a minimum.

Reactive oxygen species (ROS) play a key role in cell physiology and function. ROS represents a potential source of damage for many macromolecules including DNA. It is thought that daily changes in oxidative stress levels were an important early factor driving evolution of the circadian clock which enables organisms to predict changes in ROS levels before they actually occur and thereby optimally coordinate survival strategies. It is clear that ROS, at relatively low levels, can serve as an important signaling molecule and also serves as a key regulator of gene expression. Therefore, the mechanisms that have evolved to survive or harness these effects of ROS are ancient evolutionary adaptations that are tightly interconnected with most aspects of cellular physiology. Our understanding of these mechanisms has been mainly based on studies using a relatively small group of genetic models. However, we know comparatively little about how these mechanisms are conserved or have adapted during evolution under different environmental conditions. In this review, we describe recent work that has revealed significant species-specific differences in the gene expression response to ROS by exploring diverse organisms. This evidence supports the notion that during evolution, rather than being highly conserved, there is inherent plasticity in the molecular mechanisms responding to oxidative stress.

P63, a protein related to the tumor suppressor p53, is a transcription factor that plays an important role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (ΔN) a transactivation domain. In addition, alternative splicing at the 3' end generates proteins with different C-termini, denoted α, β and γ for a total of six isoforms. ΔNp63α isoform is the main isoform expressed at all stages of development, however the relative contribution of individual p63 isoform during ectodermal differentiation and organogenesis is still far from understood. Overexpression of ΔNp63 led to increased growth of transformed cells in vitro and in vivo while treatment of keratinocytes with ultraviolet irradiation causes down-regulation of ΔNp63 proteins and their corresponding mRNA. The p63 gene locus is often amplified in squamous cell carcinomas while alterations in the relative levels of TA and ΔNp63 correlate with prognosis in several human cancers suggesting that fine regulation of p63 intracellular levels must be of pivotal importance in controlling cell proliferation, death and differentiation. Despite its relevance little is known on the mechanisms controlling p63 protein levels. Here we show that Itch/AIP4, a HECT E3-ubiquitin ligase, promotes p63 degradation. Using a set of p63 deletion mutants, we have identified a region and two critical lysine residues of p63, associated to human Split-Hand and Foot Malformation-4 (SHFM-4) syndrome, which are involved in the mechanism of Itch-mediated p63 degradation.