The roles of iron in health and disease.

Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining body's iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.

Functional regulation of immunoproteasomes and transporter associated with antigen processing.

The central event in the cellular immune response to invading pathogens is the presentation of non-self antigenic peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). As peptide binding and transport proteins, MHC class I molecules have evolved distinct biochemical and cellular strategies for acquiring antigenic peptides, providing CTLs an extracellular representation of the intracellular antigen content. Whereas efficient generation of MHC class I binding peptides depends on the intracellular, immunoproteasome-mediated proteolysis machinery, translocation of peptides into the lumen of the endoplasmic reticulum requires the endoplasmic reticulum-resident, adenosine 5'-triphosphate (ATP) binding cassette transporter associated with antigen processing (TAP). Here we show, for the first time, that immunoproteasomes, TAP complexes, and MHC class I molecules are physically associated, providing an effective means of transporting MHC class I binding peptides from their sites of generation into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. In this review, we assess the current understanding of the functional regulation of immunoproteasomes and transporter associated with antigen processing.

Two leaky-late HSV-1 promoters differ significantly in structural architecture.

The HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (gamma1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 promoter includes only an Sp1 site at -48, a TATA box at -30, and an initiator (Inr) element at the cap site, here we show that elements upstream of -48 can functionally compensate for the mutational loss of the critical Sp1 site at -48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5' boundary of the VP16 promoter extends to ca. -90. This region includes an Sp1 binding site at -46, CAAT box homology at -77, and "E box" (CACGTG) at -85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an initiator element at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 transcription factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity.

The exchange of cognate TATA boxes results in a corresponding change in the strength of two HSV-1 early promoters.

Previous analysis of two Herpes Simplex Virus Type-1 (HSV-1) promoters controlling expression of mRNA encoding early genes (U(L)37 and U(L)50) showed that the U(L)50 (dUTPase) promoter is at least 6-fold stronger both in its normal genomic location and in the non-essential gC locus. In the present report we demonstrate that the TATA element of either promoter is the major determinant of promoter strength. When the U(L)37 TATA element (CGTATAAC) was mutated with two base changes to the U(L)50 TATA sequence (CATAAAAC) in recombinant viruses, the activity of the U(L)37 promoter was increased to that of the U(L)50 promoter. Conversely, when the U(L)50 TATA element was changed to that of the U(L)37 promoter, U(L)50 promoter activity was reduced to the level of the U(L)37 promoter. In addition, we investigated the spacing of the TATA box with respect to upstream promoter elements. We found that re-positioning the U(L)37 TATA box to a location equivalent to that of the U(L)50 promoter relative to the transcript start site; i.e. three bases upstream of its cognate location, significantly diminished activity. Substitution of the U(L)50 TATA box at the new position could only partially restore promoter activity. Thus, we also conclude that the spacing of TATA elements vis-à-vis upstream promoter elements is also a critical determinant of promoter strength.

The kinetics of VP5 mRNA expression is not critical for viral replication in cultured cells.

We generated recombinant viruses in which the kinetics of expression of the leaky-late VP5 mRNA was altered. We then analyzed the effect of such alterations on viral replication in cultured cells. The VP5 promoter and leader sequences from positions -36 to +20, containing the TATA box and an initiator element, were deleted and replaced with a strong early (dUTPase), an equal-strength leaky-late (VP16), or a strict-late (U(L)38) promoter. We found that recombinant viruses containing the dUTPase promoter inserted in the VP5 locus expressed VP5-encoding mRNA with early kinetics, while virus with the U(L)38 promoter inserted expressed such mRNA with strict-late kinetics. Further, in spite of differences in its functional architecture, the VP16 promoter fully substituted for the VP5 promoter. Western blot analysis demonstrated that the amounts of VP5 capsid protein produced by the recombinant viruses differed somewhat; however, on complementing C32 and noncomplementing Vero cells, such viruses replicated to titers equivalent to those of the rescued wild-type virus controls. Multistep virus growth in mouse embryo fibroblasts, rabbit skin cells, and Vero cells also demonstrated equivalent replication efficiencies for both recombinant and wild-type viruses. Further, recombinant viruses did not show any impairment in their ability to replicate on serum-starved or quiescent human lung fibroblasts. We conclude that the kinetics of the essential VP5 mRNA expression is not critical for viral replication in cultured cells.

Analysis of factors influencing kinetics of herpes simplex virus transcription utilizing recombinant virus.

The herpes simplex virus type 1 (HSV-1) transcription program is a regulated cascade in which early and late phases of gene expression are separated by viral DNA replication. While promoters controlling expression of transcripts encoding immediate-early proteins contain virus-specific cis-acting elements, these are in the context of cellular promoter elements, and the promoters controlling expression of other viral transcripts contain only cellular cis-acting elements. We had developed and continue to refine a general method for the production of recombinant viruses in which modified promoters can be inserted into nonessential loci within the viral genome through homologous recombination. This approach has been especially useful in defining the features of model promoters of the various kinetic classes. Our work suggests that class-specific differences in promoter architecture are critical factors in the ability of the cellular transcription machinery to form stable preinitiation complexes at various phases of infection and, thus, mediate kinetic class-specific transcription. Early (beta) promoters contain a TATA box and upstream activation elements while sequences downstream of the TATA homology are dispensible for transcription. Late transcripts can be catagorized as either leaky-late (beta gamma) or strict late (gamma) depending on whether they are readily detectable prior to viral DNA replication. Promoters controlling both types are clearly distinct from early ones in that sequences near the transcription start site which resemble consensus mammalian initiator elements are required along with the TATA box and activator elements. Strict late promoters do not contain elements upstream of the TATA box but include what appears to be a class specific element downstream of the transcription start site.