No abstract

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.

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624 -630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by ␣-helices and lies on a surface consisting of antiparallel ␤-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.Specific protective immunity results when heat shock protein (HSP) 1 -peptide complexes purified from tumor cells are injected into inbred mice bearing the same specific type of tumor (1-6). The HSP-mediated specific immunogenicity is also seen with virus-infected cells (7-9). This paradigm is the basis for a new therapeutic strategy against human cancers (9, 10). The immune response is directed against peptides noncovalently bound to the HSP and not to the HSP (for reviews, see Refs. 11 and 12)). HSPs that form the immunogenic peptide complexes include gp96 (GRP94) (13) and calreticulin (14), which reside in the endoplasmic reticulum (ER). Cytosolic HSP70-(15, 16) and HSP90-(17) peptide complexes are also immunogenic. The ER-resident chaperone gp96 (GRP94) has been the most extensively studied from an immunological standpoint (13, 18 -21). It is an abundant stress protein that displays dual functionality: it directs peptides into the immune response pathway and it assists in protein folding. The role of gp96 in the immune response is not well understood at the molecular level. gp96 binds a variety of peptides in vitro and in vivo with little or no apparent amino acid sequence specificity (13,(22)(23)(24). gp96 also binds ATP but the role of nucleotide in peptide loading/unloading is unclear (22,25). Highly purified HSP90, the cytosolic paralog of gp96, probably binds and uses ATP (26). This is in contrast to chaperones HSP70 and BiP (ER paralog of HSP70), where it is clear that ATP binding is important for the release of peptide substrates (r...

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 z-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult a-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain z-subunits and exchange with added b-subunits in the form of b 4 (HbH) significantly faster than do those with a-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 (z 2 b 2 ) < Hb Portland-1 (z 2 g 2 ) ffi Hb Gower-1 (z 2 e 2 ) < Hb Gower-2 (a 2 e 2 ) < HbF 1 < HbF (a 2 g 2 ) < HbA 2 (a 2 d 2 ), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomermonomer 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.

An 87% identity has been found between the reported cDNA sequence that encodes acylpeptide hydrolase (EC 3.4.19.1) [Mitta, M., Asada, K., Uchimura, Y., Kimizuka, F., Kato, I., Sakiyama, F. & Tsunasawa, S. (1989) J. Biochem. 106, 548-551] and a cDNA transcribed from a locus (DNFISS2) on the short arm ofhuman chromosome 3, reported by Naylor et al. [Naylor, S. L., Marshall, A., Hensel, C., Martinez, P. F., Holley, B. & Sakaguchi, A. Y. (1989) Genomics 4,[355][356][357][358][359][360][361]; the DNF15S2 locus suffers deletions in small cell lung carcinoma associated with a reduction or loss of acylase activity (EC 3.5.1.14). Acylpeptide hydrolase catalyzes the hydrolysis of the terminal acetylated amino acid preferentially from small acetylated peptides. The acetylamino acid formed by acylpeptide hydrolase is further processed to acetate and a free amino acid by an acylase. The substrates for the acylpeptide hydrolase and the acylase behave in a reciprocal manner since acylpeptide hydrolase binds but does not process acetylamino acids and the acylase binds acetylpeptides but does not hydrolyze them; however, the two enzymes share the same specificity for the acyl group. These findings indicate some common functional features in the protein structures of these two enzymes. Since the gene coding for acylpeptide hydrolase is within the same region of human chromosome 3 (3p2l) that codes for the acylase and deletions at this locus are also associated with a decrease in acylase activity, there is a close genetic relationship between the two enzymes. There could also be a relationship between the expression of these two enzymes and acetylated peptide growth factors in some carcinomas.The various types of exopeptidases that act on the free NH2-terminal residues of polypeptides have been described in detail (1). The properties of a purified enzyme that cleaves an acetylated terminal amino acid from acetylated peptides (N-acylaminoacylpeptide hydrolase, EC 3.4.19.1, referred to here as acylpeptide hydrolase) have also been reported (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). For example, this enzyme catalyzes the hydrolysis of acetyltrialanine (Ac-Ala3-OH) to acetylalanine (Ac-Ala-OH) and dialanine (Ala2-OH). We reported that the rates of hydrolysis of different blocked peptide substrates varied considerably, depending on the nature of the first and second amino acids. Thus, there was a preference for Ac-Ala-, Ac-Met-, and Ac-Ser-at the blocked terminus (4). Comparison ofthis specificity with the sequences ofabout 100 known proteins acetylated at their NH2-terminal residues indicated that most of them began with Ac-Ala-, Ac-Met-, or Ac-Ser-(13). Furthermore, in these blocked proteins there was a preponderance of charged amino acid residues at the second position. Hence, the characteristics of the terminal sequence of these blocked proteins appear to resemble the substrate specificity ofthe acylpeptide hydrolase. The enzyme displays a broad spectrum with respect to the blocking group, since acetyl, chloroacetyl, formyl,...

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 of n = 2.2 for the modified hemoglobin. After removal of unmodified Hb chains, the mixture of randomly modified acetylated alpha or beta 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 alpha-chain (Val-1 and Lys-99) and three amino groups of the beta-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(alpha), the N-terminus of the alpha-chain, and two sites between the 2 beta-chains of bovine hemoglobin, Met-1(beta) and Lys-81(beta); these findings support the conclusions of the random modification approach. Two other chloride-binding sites, Lys-99(alpha) and Lys-103(beta), 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.

The N-terminal 18-amino acid sequence of the ␤-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the ␥-chain of fetal hemoglobin with the remaining sequence of the ␤-chain retained (helices B through H). The ␥-␤-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the ␣-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramerdimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. The amino acid sequence of a protein contains the information necessary for its proper folding into the native state (1). Predicting patterns of protein folding into secondary and tertiary structures based on the primary structure is currently the focus of intense study in many laboratories. Assembly of such folded polypeptides into specific quaternary structures depends on how the individual subunits interact with one another. In human tetrameric hemoglobins there are two quaternary conformations, oxy (R) and deoxy (T), which are responsible for its functional binding and release of O 2 , respectively (2, 3). We describe here how some of these quaternary subunit interactions involving ␣ subunits in adult (␣ 2 ␤ 2 ) and fetal (␣ 2 ␥ 2 ) hemoglobins are influenced by the N-terminal sequences of their ␤ and ␥ subunits.Adult and fetal hemoglobins have several significant differences in their functional behavior at the quaternary level. These differences include their responses to DPG 1 binding, which affect their ability to carry O 2 (4), the strengths of their tetramer-dimer interactions as recently reported (5), and the circular dichroism and absorption spectra of the aromatic amino acids in their polypeptide chains (6). These well defined and accurately measured properties in adult and fetal hemoglobins are due to unique subunit interactions in the ␣ 2 ␤ 2 and the ␣ 2 ␥ 2 tetramer, respectively, that originate from the 39 amino acid substitutions between the ␤-and ␥-chains. Therefore, comparative studies of these hemoglobins are potentially informative in establishing a relationship between amino acid sequence and quaternary conformational properties.The tetramer-dimer interface of fetal hemoglobin undergoes dissociation about 70 times less readily than the corresponding interface in adult hemoglobin (5). In an effort to explain thi...

A baby girl presented with symptomatic sickle cell disease exacerbated by mild hypoxemia, despite a newborn-screening diagnosis of sickle cell trait. DNA sequencing of the beta globin gene revealed that her maternal beta globin allele was normal. Her paternal allele had not only the expected sickle-trait mutation, betaGlu6Val, but also a second, charge-neutral mutation, betaLeu68Phe. Analysis of the patient's hemoglobin revealed that the double-mutant protein, which we called "hemoglobin Jamaica Plain," had severely reduced oxygen affinity. Structural modeling suggested destabilization of the oxy conformation as a molecular mechanism for sickling in a heterozygote at an ambient partial pressure of oxygen.