An integrated analysis of herpes virus infections from eight randomized clinical studies of baricitinib in adults with moderate-to-severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is associated with an increased risk for viral infections including those caused by herpes simplex virus and varicella zoster virus. OBJECTIVES: This study examined treatment-emergent (TE) herpes simplex infection including eczema herpeticum (EH), and herpes zoster (HZ), in adult patients with AD receiving ≥1 dose of baricitinib (BARI), an oral selective inhibitor of Janus kinase 1/2. METHODS: We evaluated data from six double-blinded, randomized, placebo-controlled (PC) trials and two long-term extension studies, within three analysis sets: PC, 2-4-mg BARI extended and All-BARI-AD. Frequency, incidence rate (IR)/100 person-years (PYs) and clinical characteristics of TE-herpes simplex, EH and HZ were reported. RESULTS: In the All-BARI-AD dataset (n = 2531; 2247 PYs), herpes simplex was reported in 8.9% of patients (n = 224; IR = 10.3). Most herpes simplex events were rated as mild or moderate (93.3%), rarely led to permanent discontinuation (2.2%) and presented mostly as oral/perioral herpes simplex (51.3%). TE-EH occurred at a low frequency (All-BARI-AD 1.7% n = 43; IR = 2.0) and were reported in 0.5%, 0.2% and 1.4% of patients receiving placebo, 2-mg or 4-mg BARI respectively. In the All-BARI-AD dataset, most events were investigator-rated as mild/moderate (79.1%), affected ≤2% of the body surface area (74.2%) and occurred as single events (88.4%). Serious TE-EH (n = 11) occurred exclusively in patients with poor disease control (vIGA-AD™ score ≥3) at infection onset. TE-HZ was reported in 2.1% of BARI patients (n = 53; IR = 2.3), without a dose relationship during the PC period (IR = 2.7 and IR = 0.0) or the extended dataset (IR = 3.7 and IR = 1.7) for 2- or 4-mg BARI respectively. CONCLUSIONS: TE-herpes simplex was common, while occurrence of EH was uncommon. Most events of EH were localized with involvement of a small BSA and were linked to poor disease control. Events of HZ were rare in the PC dataset and without a dose dependent increase in frequency.

Baricitinib in patients with moderate-to-severe atopic dermatitis and inadequate response to topical corticosteroids: results from two randomized monotherapy phase III trials.

BACKGROUND: Baricitinib, an oral selective Janus kinase 1 and 2 inhibitor, effectively reduced atopic dermatitis (AD) severity in a phase II study with concomitant topical corticosteroids. OBJECTIVES: To evaluate the efficacy and safety of baricitinib in patients with moderate-to-severe AD who had an inadequate response to topical therapies. METHODS: In two independent, multicentre, double-blind, phase III monotherapy trials, BREEZE-AD1 and BREEZE-AD2, adults with moderate-to-severe AD were randomized 2 : 1 : 1 : 1 to once-daily placebo, baricitinib 1 mg, 2 mg, or 4 mg for 16 weeks. RESULTS: At week 16, more patients achieved the primary end point of Validated Investigator's Global Assessment of AD (0, 1) on baricitinib 4 mg and 2 mg compared with placebo in BREEZE-AD1 [N = 624; baricitinib 4 mg 16·8% (P < 0·001), 2 mg 11·4% (P < 0·05), 1 mg 11·8% (P < 0·05), placebo 4·8%], and BREEZE-AD2 [N = 615; baricitinib 4 mg 13·8% (P = 0·001), 2 mg 10·6% (P < 0·05), 1 mg 8·8% (P = 0·085), placebo 4·5%]. Improvement in itch was achieved as early as week 1 for 4 mg and week 2 for 2 mg. Improvements in night-time awakenings, skin pain and quality-of-life measures were observed by week 1 for both 4 mg and 2 mg (P ≤ 0·05, all comparisons). The most common adverse events in patients treated with baricitinib were nasopharyngitis and headache. No cardiovascular events, venous thromboembolism, gastrointestinal perforation, significant haematological changes, or death were observed with any baricitinib dosage. CONCLUSIONS: Baricitinib improved clinical signs and symptoms in patients with moderate-to-severe AD within 16 weeks of treatment and induced rapid reduction of itch. The safety profile remained consistent with prior findings from baricitinib clinical development in AD, with no new safety concerns.

Transferring from clopidogrel loading dose to prasugrel loading dose in acute coronary syndrome patients. High on-treatment platelet reactivity analysis of the TRIPLET trial.

High on-treatment platelet reactivity (HPR) has been identified as an independent risk factor for ischaemic events. The randomised, double-blind, TRIPLET trial included a pre-defined comparison of HPR in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) following a placebo/600-mg clopidogrel loading dose (LD) immediately before a subsequent prasugrel 60-mg or 30-mg LD. Platelet reactivity was assessed using the VerifyNow® P2Y12 assay (P2Y12 Reaction Units, PRU) within 24 hours (h) following the placebo/clopidogrel LD (immediately prior to prasugrel LD), and at 2, 6, 24, 72 h following prasugrel LDs. The impact of CYP2C19 predicted metaboliser phenotype (extensive metabolisers [EM] and reduced metabolisers [RM]) on HPR status was also assessed. HPR (PRU ≥240) following the clopidogrel LD (prior to the prasugrel LD) was 58.5% in the combined clopidogrel LD groups. No significant difference was noted when stratified by time between the clopidogrel and prasugrel LDs (≤6 hs vs>6 h). At 6 h following the 2nd loading dose in the combined prasugrel LD groups, HPR was 7.1%, with 0% HPR by 72 h. There was no significant effect of CYP2C19 genotype on pharmacodynamic (PD) response following either prasugrel LD treatments at any time point, regardless of whether it was preceded by a clopidogrel 600-mg LD. In conclusion, in this study, patients with ACS intended for PCI showed a high prevalence of HPR after clopidogrel 600-mg LD regardless of metaboliser status. When prasugrel LD was added, HPR decreased substantially by 6 h, and was not seen by 72 h.

Interferon-gamma upregulates MUC1 expression in haematopoietic and epithelial cancer cell lines, an effect associated with MUC1 mRNA induction.

Epithelial mucin-1 (MUC1) is an important target antigen that it is overexpressed in both epithelial and haematological cancers including multiple myeloma (MM) and some lymphomas and leukaemias. MUC1 has adhesive and immunosuppressive properties, which may promote cancer progression. These studies evaluated the effect of IFNs on MUC1 expression, since these agents are widely used in clinical cancer therapy. MUC1 and interferon (IFN) receptor expression were measured by radioligand binding. Changes in MUC1 mRNA levels in response to IFN-gamma were assessed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). IFN-gamma was found to be a more potent inducer of MUC1 expression than IFN-alpha. 125I-IFN binding studies indicated that both IFN receptors were expressed in most of the cell lines. With IFN-gamma treatment, there was upregulation of MUC1 mRNA. IFN-gamma has a more consistent and more potent effect upon MUC1 induction than IFN-alpha. The ability to upregulate MUC1 across a broad range of cancer types by a clinically available cytokine, IFN-gamma, has important implications for enhancing immunotherapeutic approaches targeting MUC1.

Therapeutic advantage of (90)yttrium- versus (131)iodine-labeled PAM4 antibody in experimental pancreatic cancer.

OBJECTIVES: Radioimmunotherapy studies using (131)I-PAM4 have demonstrated significant antitumor effects in mice bearing human pancreatic cancer xenografts. For several reasons (90)Y has been proposed as a more effective radionuclide for radioimmunotherapy of pancreatic cancer. The present study examined whether one radionuclide was more efficacious than the other in tumor-bearing mice. METHODS: Athymic nude mice bearing CaPan1 xenograft tumors ( approximately 1.0 cm(3)) were given increasing doses of either (90)Y-PAM4 or (131)I-PAM4 up to their respective maximal tolerated doses [MTDs (260 and 700 microCi, respectively)]. RESULTS: (90)Y-PAM4 provided significantly greater growth inhibition than the (131)I-PAM4 (P < 0.035). Median survival time for the untreated mice was 6 weeks, whereas median survival times for the (131)I-treated mice and (90)Y-treated mice at their respective MTDs were 17.5 weeks and >26 weeks (the end of the study period), respectively. Within the (131)I-PAM4-treated group, two of eight mice were responders (>50% decrease in tumor size) for a median of 14 weeks. At the end of the study (26 weeks), 1 mouse was alive with no sign of tumor. All of the (90)Y-PAM4-treated mice were responders with a median duration of response of 20 weeks. Six of the seven mice were alive at week 26, with four mice having no evidence of disease. CONCLUSIONS: These data demonstrate the advantage of (90)Y over (131)I as the radionuclide for PAM4-targeted radioimmunotherapy of xenografted pancreatic cancer. Furthermore, the duration and extent of the antitumor response suggests that multiple treatment cycles of (90)Y-PAM4 may provide an effective therapeutic for the control of pancreatic cancer.

Requirements of Cyc2p and the porin, Por1p, for ionic stability and mitochondrial integrity in Saccharomyces cerevisiae.

It was previously demonstrated that Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein; that the cyc2-Delta2 deletion lacking the entire gene causes a diminution to only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c; that the deletion causes a defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources; and that this defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a nonionic compound. We report that por1-Delta strains lacking the mitochondrial porin, Por1p, but not por2-Delta strains lacking the related porin, share some phenotypes similar to the cyc2-Delta2 strain, including hypersensitivity to KCl in glycerol medium. Moreover, spontaneous swelling in the presence of ATP was detected in mitochondria from the cyc2-Delta2 strain, while swelling could be detected in mitochondria from the other strains only after the addition of KCl. Thus, highly unspecific membrane permeation may be triggered by ATP in the cyc2-Delta2 strain. We suggest that Por1p and Cyc2p, in addition to their own unique functions, serve to maintain the osmotic stability of mitochondria, but by different mechanisms.

Localization of pancreatic cancer with radiolabeled monoclonal antibody PAM4.

Experimental animal studies were performed with (111)In-labeled PAM4 anti-MUC1 antibody along with (111)In-labeled control antibody. Tumor uptake of radiolabeled PAM4 was significantly higher than for the control antibody at all time points. When normalized to a blood dose of 1500 cGy as an estimate of myelotoxicity, (90)Y-labeled PAM4 would provide 5344 cGy to the tumor, whereas an equitoxic dose of (90)Y-labeled control antibody would provide only 862 cGy to the tumor. In addition to the animal studies, five patients with proven pancreatic cancer were administered either (131)I-PAM4 IgG (n=2) or 99mTc-PAM4 Fab' (n=3). Tumor targeting was observed in four out of five patients. By immunohistochemistry, PAM4 was non-reactive with tumor from the one patient not targeted. Dosimetry from the patients given (131)I-PAM4 predicted that tumors would receive 10-20 cGy/mCi with tumor/red marrow dose ratios ranging from 3 to 10. Based upon these results, we have established a phase-I (111)In-labeled PAM4 imaging and (90)Y-labeled PAM4 therapy trial.

Monoclonal antibody G47 engineered to be reactive with colorectal tumor mucin.

We have previously shown that the normal adult colon produces a sialomucin containing the core trisaccharide 1,3 N-acetylgalactosamine. This structure was shown to be the epitope for a polyclonal antiserum that demonstrated colon "specific" activity. Antiserum binding is dependent upon the presence of O-acetylated sialic acids present at high concentrations in normal adult colon tissue. However, O-acetylation of sialic acids is decreased in colorectal cancer. Indeed, approximately 50% of colorectal carcinomas are nonreactive with this antiserum. In the current work, we used a de-O-acetylated, normal colon mucin as immunogen to generate monoclonal antibody (MAb) G47. Untreated normal colon mucins having a high O-acetylated sialic acid content were essentially nonreactive with G47. Removal of O-acetyl groups by saponification generated a reactive mucin derivative while subsequent treatment with neuraminidase abolished reactivity. By immunoperoxidase procedures MAb-G47 was reactive with approximately 85% of colorectal tumors while exhibiting relatively low reactivity with normal colon tissue. Mucins isolated from normal colon had on average less than 10% of the specific epitope as compared with mucins derived from colorectal tumors (p < 0.01). Initial immunohistochemical studies on tumors of noncolonic origin revealed few positive cases. The potential of MAb-G47 to assist in the diagnosis and/or prognosis of colorectal cancer is now being studied.

Epithelial mucin-1 (MUC1) expression and MA5 anti-MUC1 monoclonal antibody targeting in multiple myeloma.

Multiple myeloma (MM) is the second most common hematological cancer in the United States. It is typically incurable, even with myeloablative chemotherapy and stem-cell transplantation. The epithelial mucin-1 (MUC1) glycoprotein is expressed by normal and malignant epithelial cells but has also been shown to be expressed by MM cells. MUC1 is a useful antigenic target in solid tumors for clinical diagnostic and therapeutic monoclonal antibody (mAb)-based approaches. The MA5 mAb, as well as other anti-MUC1 mAbs reactive with the MUC1 variable number tandem repeat domain, exhibited moderate to strong reactivity with both MM cell lines and clinical samples. To explore the biochemical nature and potential of MUC1 as an antigenic target in MM, studies were performed to: (a) compare the mRNA and the MUC1 glycoprotein species between epithelial cancer and MM cell lines; and (b) develop and use a human MM tumor xenograft model system to study the biodistribution of the MA5 mAb. MA5 mAb was strongly reactive with six of eight human MM cell lines by flow cytometry. In seven of eight MM patient samples (bone marrow and/or peripheral blood) reactivity was found in 10-90% of the cells, whereas normal control (n = 5) and leukemia and lymphoma (n = 5) cells showed only 0-6% reactivity. 125I-labeled MA5 whole-cell binding studies showed quantitatively similar amounts of binding between strongly positive MM lines and high-MUC1-expressing breast carcinoma lines. mRNA expression was assessed by Northern blotting and reverse transcription-PCR. MM cell lines were positive by both methods, with strong similarity in the sizes of the mRNAs and cDNAs that were obtained. Finally, biodistribution experiments were carried out with 131I-labeled MA5 versus a nonbinding control 125I-labeled mAb in a s.c. MM xenograft model. Selective MM tumor uptake of the MA5 mAb was demonstrated, with a potential for delivering a tumor radiation absorbed dose of 8540 cGy/mCi of injected dose compared with 3099 cGy/mCi of tumor-absorbed dose delivered by nonspecific antibody.

Cyc2p is required for maintaining ionic stability and efficient cytochrome c import and mitochondrial function in Saccharomyces cerevisiae.

A previous study demonstrated that. Cyc2p from Saccharomyces cerevisiae is a mitochondrial protein and that cyc2 mutants contained only approximately 20% of the normal levels of cytochrome c due to a partial deficiency in mitochondrial import of apo-cytochrome c. We report herein that deletion of the entire gene results in defective mitochondrial function, as revealed by diminished growth on media containing nonfermentable carbon sources. This defect is exacerbated in hyper-ionic KCl media and at higher incubation temperatures, but is suppressed on media containing sorbitol, a non-ionic compound. We suggest that Cyc2p serves to maintain the osmotic stability of mitochondria, and its defect is exacerbated by KCl.

Analysis of mutationally altered forms of the Cct6 subunit of the chaperonin from Saccharomyces cerevisiae.

The Cct double-ring chaperonin complex of Saccharomyces cerevisiae is comprised of eight essential subunits, Cct1p-Cct8p, and assists the folding of substrates such as actins and tubulins. Single and multiple amino acid replacements of Cct6p were constructed by oligonucleotide-directed mutagenesis, including changes of charged to alanine residues and uncharged to charged residues. The replacements were targeted, in part, to residues corresponding to functionally critical regions identified in the published crystal structure of the Escherichia coli chaperonin, GroEL. Here, we report the critical hydrophobic residues and clusters of hydrophilic residues in regions corresponding to those from the apical domain of GroEL implicated in peptide binding and peptide release, and certain residues in the putative equatorial domain implicated in subunit-to-subunit interaction. In contrast to their homologous counterparts in Cct2p and Cct1p, the highly conserved putative ATP binding motifs of Cct6p were relatively amenable to mutations. Our data suggest that the entire Cct6p molecule might be essential for assembly of Cct complex and might participate in binding substrates. However, there appeared to exist a functional hierarchy in ATP binding/hydrolysis among Cct subunits, as suggested by the high tolerance of Cct6p to mutations within the putative ATP binding pocket.

Radioimmunotherapy of experimental pancreatic cancer with 131I-labeled monoclonal antibody PAM4.

We examined the therapeutic efficacy of 131I-labeled murine monoclonal antibody (MAb) PAM4 against human pancreatic cancers carried as xenografts in athymic nude mice. Animals bearing the CaPan1 tumor (0.2 cm3) were either untreated or were given, 131I-labeled nonspecific Ag8 antibody. By week 7 mean tumor size had grown 16.5 +/- 8.4-fold and 4.2 +/- 2.5-fold for the untreated and 131I-Ag8-treated animals, respectively. In contrast, animals administered 131I-PAM4 exhibited marked regression of tumors to an average of 15% of initial tumor volume. Since most pancreatic cancer patients present with large tumor burdens, the limitation of 131I-PAM4 treatment with respect to initial tumor size was investigated in animals bearing tumors of approximately 0.5 cm3, 1.0 cm3 and 2.0 cm3. Significant extension of survival time (>3-fold increase) was noted for both the 0.5 cm3 and 1.0 cm3 131I-PAM4-treated groups, compared to their respective untreated controls. Even in the group bearing large 2.0-cm3 tumors, survival was increased 2-fold over the control group. To further improve anti-tumor effects in large tumors, 2 injections of 131I-PAM4 were administered at a 4-week interval to animals bearing tumors of approximately 1.0 cm3. Significant extended survival was noted for the group receiving 2 doses when compared to the group receiving only 1 dose.

A phase II trial of deferoxamine in patients with hormone-refractory metastatic prostate cancer.

The management of hormone-refractory metastatic prostate cancer remains a therapeutic dilemma. We report the results of a phase II trial with deferoxamine administrated at a dose of 50 mg/kg (maximum dose 5 g) administered intravenously over 8 hr daily, repeated for 5 days at 4-week intervals for 2 courses. Fourteen patients with advanced hormone-refractory prostate cancer were treated and 28 courses were delivered. Essentially no toxicity was observed. Using combined clinical and prostate-specific antigen (PSA) criteria. 13 of 14 patients had disease progression. However, 9 of 14 patients had stable measurable or evaluable disease and progressed solely based on PSA criteria. Deferoxamine in this dose and schedule has no activity in hormone-refractory prostate cancer. Further investigation of the effect of deferoxamine on PSA production/expression is warranted.

Inhibition of lymphoma growth in vivo by combined treatment with hydroxyethyl starch deferoxamine conjugate and IgG monoclonal antibodies against the transferrin receptor.

Synergistic inhibition of hematopoietic tumor growth can be observed in vitro when the iron chelator deferoxamine (DFO) is used in combination with an IgG mAb against the anti-transferrin receptor antibody (ATRA). Our goal was to ascertain whether similar findings could be seen in vivo. A high molecular weight conjugate of deferoxamine, known as hydroxyethyl starch (HES) DFO or HES-DFO, was tested in conjunction with C2, a well-defined rat antimouse transferrin receptor mAb, against the 38C13 tumor in C3H/HeN mice. It was shown that while neither HES-DFO alone nor C2 alone produced consistent, significant inhibition of tumor growth, the combination of HES-DFO and C2 produced virtually complete inhibition of initial tumor outgrowth. The latter combination failed, however, to inhibit the growth of established tumors. It was then found that when C2 was used in conjunction with RL34, another IgG ATRA, the two ATRAS were themselves capable of causing synergistic inhibition of the growth of 38C13 in vitro. When the two IgG ATRAS were used together in vivo, regressions of established tumors were observed. Moreover, the addition of HES-DFO to the IgG ATRA pair then caused more frequent regressions. Although there was never any obvious toxicity seen with a single IgG ATRA, the use of the IgG ATRA pair was associated with sporadic mortality. In addition, although HES-DFO by itself was also not associated with any obvious toxicity, combined treatment with HES-DFO and a single ATRA resulted in death due to bacterial infection in about half of the mice after 10-15 days. Combined treatment with HES-DFO and the ATRA pair resulted in death attributed to infection in nearly all of the mice after 6 days. Thus, an iron deprivation treatment protocol with HES-DFO and IgG ATRAS produced both a significant antitumor effect and an increased risk of infection in a murine model system.

Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast.

Members of the Hsp60 chaperonin family, such as Escherichia coli GroEL/S and the eukaryotic cytosolic chaperonin complex, TRiC (TCP ring complex), are double toroid complexes capable of assisting the folding of proteins in vitro in an ATP-dependent fashion. TRiC differs from the GroEL chaperonin in that it has a hetero rather than homo-oligomeric subunit composition and lacks a GroES-like regulatory cofactor. We have established greater than 57% identity between a protein encoded by the TCP20 gene from a human cDNA library and the newly identified protein encoded by the TCP20 gene located on the right arm of chromosome IV of the yeast Saccharomyces cerevisiae. These Tcp20 proteins showed approximately 30% identity to Tcp1, a known subunit of TRiC. Gel filtration, followed by Western analysis of purified bovine testis TRiC with a Tcp20-specific antibody, indicated that Tcp20 is a subunit of the hetero-oligomeric TRiC. Gene disruption experiments showed that TCP20, like TCP1, is an essential gene in yeast, consistent with the view that TRiC is required for folding of key proteins. The amino acid sequence similarities and the derived evolutionary relationships established that the human and yeast Tcp20 proteins represent members of a new family of subunits of TRiC chaperonins.

Characterization of monoclonal antibody PAM4 reactive with a pancreatic cancer mucin.

A monoclonal antibody (MAb), PAM4, having reactivity with pancreatic carcinoma has been developed. PAM4 is an IgG1 immunoglobulin produced by immunization of mice with mucin purified from the xenografted RIP1 human pancreatic carcinoma. An immunohistochemical study of normal adult tissues showed the PAM4 reactive epitope to be restricted to the gastrointestinal tract and absent from normal pancreas. In neoplastic tissue, PAM4 was reactive with 85% of the pancreatic carcinomas, approximately half of the colon cancers and none of the breast, ovarian, prostate, renal and liver cancers. PAM4 was, in general, non-reactive with pancreatitis specimens whereas CA19.9 and DUPAN2 were strongly reactive with each one. Treatment of the mucin antigen by heating, reduction of disulfide bonds, or protease digestion abolished immunoreactivity with PAM4. Treatment of the mucin by neuraminidase or periodate oxidation reduced immunoreactivity but did not completely abolish it. Our data are consistent with the proposal that the PAM4 epitope is a conformationally dependent peptide epitope and that certain carbohydrate structures are necessary in order to maintain the correct peptide conformation. The high specificity and intense reactivity of PAM4 with pancreatic carcinoma tissue suggests that the antibody may prove useful for in vitro diagnostic assays as well as in vivo targeting of diagnostic and therapeutic agents.

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.

Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

Enhanced thermodynamic stabilities of yeast iso-1-cytochromes c with amino acid replacements at positions 52 and 102.

We have determined the structures and thermodynamic stabilities of the wild type Asn-52 and unusually thermostable mutant Ile-52 yeast iso-1-cytochromes c (Das, G., Hickey, D. R. McLendon, D., McLendon, G., and Sherman, F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 496-499). Although both structures were similar, Water-166, buried within the wild type protein, is excluded from the Ile-52 mutant, which substantially reorganizes the local hydrogen bonding. Wild type Cys-102 was replaced with alanine or serine to eliminate dimerization in vitro. The Cys-102 (wild type), Ala-102, and Ser-102 proteins were equally stable, whereas the chemically modified Cys-102-SCH3 was less stable. The order of stability observed with replacements at positions 52 and 102 was as follows: Ile-52 Ala-102 greater than Ala-52 Ala-102 greater than Asn-52 Ala-102 ("normal") greater than Gly-52 Ala-102. No significant stabilization was attributed to potential energy interactions expressed as helix-forming propensities of replacements at position 52. A high correlation between differences in free energy changes and transfer free energies suggests hydrophobic interactions are the main factor for enhancing stability in the Ile-52 mutant. Additional possible contributions to the thermostability of the Ile-52 variant are energetic effects due to packing and hydrogen bonding changes surrounding position 52.