Anthony Popowicz

Inhibition of the G1-S transition of the cell cycle by inhibitors of deoxyhypusine hydroxylation.

The formation of the unusual amino-acid hypusine in eIF-5A (eukaryotic initiation factor 5A) is associated with cellular proliferation. We used a panel of compounds, including mimosine, to probe the relationship between the exit from the G1 phase of the cell cycle, i.e., the onset of DNA replication, and the formation of hypusine by the enzyme deoxyhypusyl hydroxylase (DOHH). These two parameters displayed the same dose dependency and structure-activity relationship. Only compounds that inhibited DOHH also suppressed proliferation. This effect was observed: (i) in spontaneously proliferating, virally transformed, and mitogen-stimulated cells; (ii) for both anchorage-dependent and anchorage-independent proliferation; and (iii) with normal and malignant cell lines. DOHH reactivation occurred rapidly after inhibitor withdrawal and correlated with synchronized entry into S. The changes in the expression of specific genes during the G1-to-S transition mimicked the physiological pattern. These findings suggest that hypusine formation in eIF-5A which occurs in a specific, invariant sequence motif acquired early in evolution, may be involved in the G1-to-S transition in the eukaryotic cells tested.

Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.

Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer–dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic ζ‐subunits leads to an average 20‐fold weakening of tetramer–dimer interfaces compared to corresponding hemoglobins containing adult α‐subunits. The dimer–monomer interfaces of these hemoglobins differ by at least 500‐fold in their strengths; such interfaces are weak if they contain ζ‐subunits and exchange with added β‐subunits in the form of β4 (HbH) significantly faster than do those with α‐subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland‐2 (ζ2β2) < Hb Portland‐1 (ζ2γ2) ≅ Hb Gower‐1 (ζ2ε2) < Hb Gower‐2 (α2ε2) < HbF1 < HbF (α2γ2) < HbA2 (α2δ2), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer–monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic → fetal → adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.

Random chemical modification of the oxygen-linked chloride-binding sites of hemoglobin: Those in the central dyad axis may influence the transition between deoxy- and oxy-hemoglobin

The features of random chemical modification are defined with reference to acetylation of bovine hemoglobin, which has been performed in a random manner so that all of the amino groups that participate in functional chloride binding (i.e., those that are oxygen-linked) could be identified. Random chemical modification, which has objectives different from those of both specific (selective) and extensive chemical modification, has been achieved for bovine hemoglobin with the mild reagent,14C-methyl acetate phosphate; retention of function was demonstrated by a Hill coefficient ofn=2.2 for the modified hemoglobin. After removal of unmodified Hb chains, the mixture of randomly modified acetylated α or β chains was subjected to tandem treatment with trypsin and chymotrypsin. Peptides were purified by HPLC and identified by amino acid analysis. The amount of radioactivity in the acetylated amino group of a purified peptide was taken as an estimate of the degree of chloride binding. For bovine Hb, two amino groups of the α-chain (Val-1 and Lys-99) and three amino groups of the β-chain (Met-1, Lys-81, and Lys-103) were shown to be oxygen-linked (i.e., to have incorporated significantly more radioactivity in the deoxy conformation compared to the same site in the oxy conformation). Three of these sites were already known chloride-binding sites [i.e., Val-1(α), the N-terminus of the α-chain, and two sites between the 2 β-chains of bovine hemoglobin, Met-1(β) and Lys-81(β); these findings support the conclusions of the random modification approach. Two other chloride-binding sites, Lys-99(α) and Lys-103(β), align the sides of the central dyad axis connecting the two well-known major chloride-binding sites of bovine Hb. The interrelationship of these five chloride-binding sites was assessed by improved molecular graphics. When viewed through the central dyad axis, the functional chloride-binding sites in the central cavity appear to be symmetrically related and to connect the two major chloride-binding sites. Modifiers or mutants that are directed at these regions in the central dyad axis may favor the deoxy conformation to provide a lower oxygen affinity by preventing the constriction of the central cavity that normally occurs upon oxygenation.

N-terminal acetylation and protonation of individual hemoglobin subunits: position-dependent effects on tetramer strength and cooperativity.

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N‐terminals of either the α‐ or the β‐subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N‐terminals of the α‐subunits of liganded hemoglobin results in a significantly increased basicity (increased pKa values) and a reduction in the strength of subunit interactions at the allosteric tetramer–dimer interface. Cooperativity in O2 binding is also decreased. Substitution of Ala at the N‐terminals of the β‐subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer–dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the α‐subunit, the slope of the plot of the tetramer–dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the α‐subunits is less extensive than that of the β‐subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

N-terminal contributions of the γ-subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70‐fold smaller tetramer‐dimer dissociation constant. This property has been shown previously to be only partially caused by the 5‐amino‐acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18‐amino‐acid N‐terminal A helix of the γ‐subunit of fetal hemoglobin, which differs from the β‐subunit of adult hemoglobin at eight amino acid residues. This long‐distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the γ‐subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the β‐subunit by the corresponding residues in the γ‐subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (γ) of HbF with its high pKa of 8.1 compared with a 7.1 value for Val 1 (β) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the γ‐subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (β) by Glu 5 (γ) promotes tetramer strengthening. The Glu → Asp replacement at position 7 enhances this effect because of the lower pKa of Asp but the Val → Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen‐binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer‐dimer allosteric interface, oxygen‐binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.

Expression and Properties of Recombinant HbA2 (α2δ2) and Hybrids Containing δ-β Sequences

Hemoglobin A2 (α2δ2), which is present at low concentration (1–2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties. The construct expressing HbA2 contains only α and δ genes so that the extensive purification required to isolate natural HbA2 is circumvented. Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant α2δ2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the α + β or the α + γ genes, respectively, are present on the construct. Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA2 are described.

REMOTE CONTRIBUTIONS TO SUBUNIT INTERACTIONS : LESSONS FROM ADULT AND FETAL HEMOGLOBINS

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INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

The expression of the six types of human Hb subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the ‘extrinsic’ component of regulation). Here, we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the ‘intrinsic’ component of regulation). The adult Hb dimers have the strongest subunit interfaces and the embryonic Hbs the weakest, with fetal Hbs being of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these Hb types form functional O2‐binding tetramers consistent with gene switching.

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces

Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low‐Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties.