Zhonghua Wang

Converting cytochrome C into a peroxidase-like metalloenzyme by molecular design.

Hemoproteins, which have a heme prosthetic group, are excellent natural models for artificial design of desired metalloproteins. Various hemoproteins can perform a range of functions by means of combining heme with a different protein scaffold, especially the coordination environment of the heme, which includes the axial ligand, coordination number, and coordination sphere. Consequently, the de novo design of a heme pocket could be a useful strategy for constructing new hemoproteins with desired or novel properties and functions. Although cytochrome c (Cyt c), an electron transfer protein, has no peroxidase activity in living systems, previous studies have shown that it can catalyze a variety of oxidation reactions in the presence of H2O2. [3] Furthermore, characterized by the covalent attachment of a heme to the polypeptide, Cyt c is a very stable protein and has several advantages for use as a peroxidase mimic. 4] In addition, it was recently discovered that the release of Cyt c from mitochondria in the initial stages of apoptosis is related to the cardiolipin oxygenase activity of the cytochrome. This provides a compelling basis for understanding the structural features of the protein that dictates the chemical reactivity of the heme. However, the intrinsic peroxidase activity of Cyt c is suppressed by the protein matrix when compared to typical peroxidases, such as horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP), which have a penta-coordination heme iron and a distal histidine in the heme pocket. Therefore, replacing the sixth axial ligand (Met80) with a non-coordination amino acid and introducing a distal histidine in the heme pocket could convert Cyt c into a peroxidase-like metalloenzyme. To achieve the above-mentioned purpose, we investigated the peroxidase activity of yeast iso-1-cytochrome c (PDB ID: 2YCC) variants in which selected substitutions at the active site had been introduced in an initial effort to mimic some of the structural features of classic peroxidases, such as HRP and CcP. Molecular modeling suggests that replacement of Tyr67 with histidine should place the Ne of His67 at ~5.2 B from the heme iron. This distance was obtained from an energy-minimized simulation of a molecular model based on the PDB file 2YCC, which was created with VMD and NAMD. This distance approximates to that between the Ne of the distal histidine and the heme iron in HRP (PDB ID: 1H5A) and CcP (PDB ID: 2CYP; 5.84 and 5.55 B, respectively). This result, combined with the lack of a coordinated axial ligand in either HRP or CcP, led us to construct and evaluate the peroxidase activity of Cyt c Met80Val, Tyr67His, and Tyr67His/Met80Val variants in which either the sixth axial ligand (Met80) was eliminated and/ or a distal histidine at position 67 was introduced in the heme pocket (Figure 1).

Cisplatin and gemcitabine as the first line therapy in metastatic triple negative breast cancer

No standard first‐line treatment exists for patients with metastatic triple‐negative breast cancer (mTNBC). In this single‐arm, phase II study (NCT00601159), we evaluated the efficacy and tolerability of cisplatin and gemcitabine (GP) as the first‐line therapy in mTNBC. Eligible women were those who had measurable disease with no prior chemotherapy for mTNBC. All patients received 21‐day‐cycle of cisplatin 25 mg/m2 on days 1–3 and gemcitabine 1,000 mg/m2 on days 1 and 8. Treatment was continued until disease progression, unacceptable toxicity or up to 8 cycles. BRCA1/2 mutation status and immunohistochemical basal markers were included in the correlative studies. Sixty‐four patients with the median age of 49 years were enrolled. Thirty patients (46.9%) had ≤1 year from diagnosis to recurrence. The median progression free survival (PFS) was 7.2 months (95%CI, 5.6–8.9 months) and overall survival (OS) was 19.1 months (95%CI, 12.4–25.8 months) with median follow‐up 42 months. Patients received treatment for a median of six cycles. The overall response rate was 62.5%. The most common grades 3/4 toxicities were neutropenia (42.2%), thrombocytopenia (29.7%), anemia (18.8%) and nausea/vomiting (15.6%).No specific BRCA1/2 mutation carriers were identified. The efficacy of responses and basal‐like subtype were independent favorable factors for PFS and OS, respectively. We conclude that the combination of GP has significant activity and a favorable safety profile as the first‐line chemotherapy in mTNBC patients, in particular patients with basal‐like subtype. The promising role of this combination as the front‐line treatment for mTNBC continued to be evaluated in our ongoing phase III trial (CBCSG006).

Evolutionary alkaline transition in human cytochrome c

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pKa value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.

Structural Basis for Cytochrome c Y67H Mutant to Function as a Peroxidase

The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe3+ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H2O2 by functioning as an acidic catalyst.

Conformational Toggling of Yeast Iso-1-Cytochrome c in the Oxidized and Reduced States

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined. The structures indicate that the half molecule of cyt c (aa 50–102) presents a kind of “zigzag riveting ruler” structure, residues at certain positions of this region such as Pro71, Lys73 can move a big distance by altering the tertiary structure while maintaining the secondary structures. This finding provides a molecular insight into conformational toggling in different oxidation states of cyt c that is principle significance to its biological functions in electron transfer and apoptosis. Structural analysis also reveals that Pro71 functions as a key hydrophobic patch in the folding of the polypeptide of the region (aa 50–102), to prevent heme pocket from the solvent.

Generation of novel functional metalloproteins via hybrids of cytochrome c and peroxidase

The continued interest in protein engineering has led to intense efforts in developing novel stable enzymes, which could not only give boost to industrial and biomedical applications, but also enhance our understanding of the structure-function relationships of proteins. We present here the generation of three hybrid proteins of cytochrome c (cyt c) and peroxidase via structure-based rational mutagenesis of cyt c. Several residues (positions 67, 70, 71 and 80) in the distal heme region of cyt c were mutated to the highly conserved amino acids in the heme pocket of peroxidases. The multiple mutants were found to exhibit high peroxidase activity and conserve the impressive stability of cyt c. We expect that this strategy could be extended to other cases of metalloprotein engineering, and lead to the development of stable and active biocatalysts for industrial uses. Besides, this study also provides insight into the structure-function relationships of hemoproteins.

A phase II trial of biweekly vinorelbine and oxaliplatin in second- or third-line metastatic triple-negative breast cancer

Patients with metastatic triple-negative breast cancer (mTNBC) typically have a poor prognosis. The purpose of this study was to prospectively evaluate the efficacy and toxicity of biweekly combination of vinorelbine and oxaliplatin (NVBOX) in second- or third-line setting for mTNBC. Eligible patients were female with 18–70 y old, and had mTNBC that had progressed after 1or 2 prior chemotherapy regimens in the metastatic setting. NVBOX was given biweekly every 4 week for a maximum of 6 cycles. The primary endpoint was progression-free survival (PFS). Forty-4 patients were recruited. All patients had been exposed to anthracyclines and/or taxanes; 56.8% of patients were cis/carbo-platin pretreated. Among the 38 evaluable patients, overall response rate was 31.6% and 7 lasted ≥ 6 months. The median PFS and overall survival (OS) were 4.3 (95% CI, 3.6–5.0) months and 12.6 (95% CI, 8.1–17.0) months, respectively. PFS and OS was significantly shorter in patients with interval from diagnosis to recurrence ≤ 1 y and time to progression (TTP) of 1–2 previous regimens before recruitment ≤ 3 months. For 34 patients who were treated in second line setting, prior platinum was a factor significantly compromising the PFS of NVBOX. Grade 3/4 hematologic toxicities included neutropenia (70.5%), thrombocytopenia (27.3%) and anemia (15.9%). The most frequent grade 3/4 non-hematologic toxicities were constipation/abdominal distension (20.5%) and nausea/vomiting (13.6%). We conclude that biweekly NVBOX regimen is effective with a good safety profile in the second- or third-line mTNBC, which warrants further investigation in a phase III study. This trial was registered with www.clinicaltrials.gov (no. NCT01528826).

A route to novel functional metalloproteins via hybrids of cytochrome P450 and cytochrome c.

One challenging topic in the field of protein engineering is to design novel functional proteins, which would provide not only a critical test of our understanding of the structure–function relationships of proteins, but also applications towards biocatalysis, pharmaceuticals and diagnostics. Metalloproteins, which account for nearly half of all proteins in nature, are particularly attractive in engineering because of their ubiquitous presence in vivo such as in biocatalysis, in energy metabolism and in signal transduction. 2] Significant advances have been made in this area and, as recently reviewed by Lu, novel metalloproteins have mainly been designed by three strategies : the de novo design of protein scaffolds, the design of metal-binding sites in native scaffolds and the design of metalloproteins with the use of unnatural amino acids or non-native metal cofactors. Recently, we have been exploring a new route to novel functional metalloproteins, involving the construction of hybrid protein scaffolds from different metalloproteins. The hybrid scaffolds contain specific motifs as a result of rational design, and the resulting proteins combine the merits of distinct metalloproteins. Here we report the construction of five hybrid proteins through hybridization of two different metalloproteins—cytochrome c (Cyt c) and cytochrome P450 (CYP450)—by genetic and protein engineering. The gene overlap extension method was employed to construct the plasmids and a high-level expression and purification system for the hybrid proteins was developed. The stabilities and catalytic activities of the hybrid proteins were examined in detail. Interestingly, all the hybrid proteins possess superior stabilities, and some of the resulting metalloproteins have acquired the epoxidation activity of CYP450. More strikingly, the peroxidase activity of one particular hybrid protein is more than 40 times greater than that of Cyt c. One of the fundamental purposes of protein engineering is to develop stable enzymes for industrial applications. In this context, Cyt c, which has a remarkable stability attributed to its compact globular structure and covalently bound heme group, could serve as an excellent structural framework in engineering. CYP450, another heme-containing protein, is one of the most versatile enzymes in nature and could potentially efficiently catalyze hundreds of different substrates through a variety of difficult biotransformations such as epoxidations and hydroxylations. However, strict reaction conditions are required for CYP450 catalysis in vitro, obstructing possible industrial applications. In CYP450, six flexible protein segments, named “substrate recognition sites” (SRSs), are considered to be the key contributors to its catalytic diversity, involved in moving, recognizing and binding substrates in an induced-fit mechanism. 4] Thus, in order to obtain stable and catalytic proteins, the SRS segments of CYP450 were introduced into the scaffold of yeast iso-1 Cyt c by replacement of the residues in its 78–85 loop, which is one of the least stable and last folding units in Cyt c. The hybrid proteins of Cyt c with CYP450s SRS-1, SRS-2, SRS-3, SRS-5 and SRS-6 were named HY1, HY2, HY3, HY5 and HY6, respectively. Firstly, we conducted molecular dynamics simulations for the rational design of hybrid proteins. Whereas Cyt c does not have any cavity around the heme pocket, according to the analysis, it is interesting to note that HY1 and HY6 each possess a cavity in the distal heme region. In HY1 the volume of the pocket is over 150 3 (Figure 1), providing a molecular basis for the binding of substrates.

The Role of Ile476 in the Structural Stability and Substrate Binding of Human Cytochrome P450 2C8

The biological function and stability of a cytochrome P450 (CYP) mainly depend on the subtle properties of the residues in the active site cavity, which are generally more divergent among proteins than other parts of the protein. As the most unique member of human CYP2C family, CYP2C8 has an isoleucine (Ile) 476 instead of phenylalanine (Phe) in substrate recognizing site 6 (SRS6). However, the role of Ile476 of CYP2C8 is still unknown. Therefore, six site-directed mutants of CYP2C8 were constructed to better define this. By UV–visible and circular dichroism spectroscopy studies, we studied for the first time the structural stability and all-trans-retinoic acid binding capability of the CYP2C8 variants. We found that the ferric CYP2C8 went through three states during thermal unfolding. Combined with substrate binding studies, our data revealed that residue 476 was involved in contact with substrate and was important for maintaining the thermal stability of CYP2C8.

Efficacy and safety of everolimus in Chinese metastatic HR positive, HER2 negative breast cancer patients: a real-world retrospective study

Background Everolimus combined with endocrine therapy has been proved to be effective among postmenopausal women with hormone receptor-positive human epidermal growth factor receptor-2 negative (HR+/HER2-) metastatic breast cancer (MBC). We aimed to evaluate the efficacy and safety of everolimus plus endocrine therapy in Chinese real-world practice for the first time, and investigate factors associated with efficacy. Methods Seventy-five HR+/HER2- MBC patients were included in this retrospective study who received everolimus plus endocrine therapy after progression on prior endocrine therapy in Fudan University Shanghai Cancer Center (FUSCC) between June 2013 and February 2016. Main outcome measures are progression free survival (PFS), overall survival (OS), objective response rate (ORR), clinical benefit rate (CBR) and safety profile. Results After a median follow up of 10.3 (range: 2.1-32.2) months, median PFS was 5.9 months (95%CI 4.6-7.2), and median OS was not reached. The CBR was 38.8% (95%CI, 26.8-50.8) and ORR was 9.0% (95%CI, 2.0-16.0). Most common all-grade adverse events were stomatitis (57.1%), fatigue (25.7%), infection (24.3%) and hyperglycemia (21.4%). The most common ≥3 grade adverse events were stomatitis (9.3 %) and thrombocytopenia (5.7%). No treatment-related death was documented during and one month after the drug administration. Conclusions The combination of everolimus and endocrine therapy proved to be effective in Chinese population. The safety profiles were similar to previous studies but incidences were lower. In conclusion, everolimus combined with endocrine therapy provides a reasonable option for Chinese HR+/HER2- metastatic breast cancer patients.BACKGROUND Everolimus combined with endocrine therapy has been proved to be effective among postmenopausal women with hormone receptor-positive human epidermal growth factor receptor-2 negative (HR+/HER2-) metastatic breast cancer (MBC). We aimed to evaluate the efficacy and safety of everolimus plus endocrine therapy in Chinese real-world practice for the first time, and investigate factors associated with efficacy. METHODS Seventy-five HR+/HER2- MBC patients were included in this retrospective study who received everolimus plus endocrine therapy after progression on prior endocrine therapy in Fudan University Shanghai Cancer Center (FUSCC) between June 2013 and February 2016. Main outcome measures are progression free survival (PFS), overall survival (OS), objective response rate (ORR), clinical benefit rate (CBR) and safety profile. RESULTS After a median follow up of 10.3 (range: 2.1-32.2) months, median PFS was 5.9 months (95%CI 4.6-7.2), and median OS was not reached. The CBR was 38.8% (95%CI, 26.8-50.8) and ORR was 9.0% (95%CI, 2.0-16.0). Most common all-grade adverse events were stomatitis (57.1%), fatigue (25.7%), infection (24.3%) and hyperglycemia (21.4%). The most common ≥3 grade adverse events were stomatitis (9.3 %) and thrombocytopenia (5.7%). No treatment-related death was documented during and one month after the drug administration. CONCLUSIONS The combination of everolimus and endocrine therapy proved to be effective in Chinese population. The safety profiles were similar to previous studies but incidences were lower. In conclusion, everolimus combined with endocrine therapy provides a reasonable option for Chinese HR+/HER2- metastatic breast cancer patients.