Brief Biography

Research in this program focuses on the study of the relationship between molecular structure and biological function of proteins and other macromolecules. The goal is to gain a better understanding of the molecular bases of disease processes.

Dr. Bibudhendra (Amu) Sarkar is an international authority on metal-caused diseases. He developed the copper-histidine treatment for Menkes disease, a devastating neurodegenerative disease in children caused by a genetic defect of copper transport which causes children to die before the age of three. The oldest patient treated by copper-histidine is a 22-year-old man. Amu has edited five books and published more than 200 scientific articles. He leads an international laboratory where he trained 60 postdoctoral fellows and graduate students from all over the world.

He is a graduate of the University of Southern California, Los Angeles, with a PhD in Biochemistry. During his 38-year service at SidkKids, he received numerous honours and awards including being the first recipient of Medical Research Council Scholar Award at SickKids, Nuffield Foundation Award of UK, Visiting Professorships in the Universities of Cambridge and Paris, and had delivered an invited lecture before the Nobel Symposium under the auspices of the Nobel Foundation in Sweden for his pioneering research in inorganic bio-chemistry.

The following article was published in IUBMB Life and has been reproduced with permission of the publisher Taylor & Francis Group.

How I became a biochemist by Bibudhendra Sarkar, IUBMB Life, (2003) vol. 55 no. 4-5 pp 287-289.

How I became a biochemist by Bibudhendra Sarkar
[Adobe Acrobat PDF 121 KB]

Research Interests

• Metalloproteomics
• Studies of copper-transporting ATPases
• Treatment developments for Menkes and Wilson diseases
• Heavy metals in the environment

Research Activities

1. Metalloproteomics:

Mammalian systems are too complex to be deciphered by their genes alone. Genomic data and transcript profiling offer opportunities to identify molecular alteration in disease, but they do not specify which specific proteins interact, how these proteins occur or how long they persist in a biological system. Proteomics, the global study of proteins, encompasses protein expression and structure-function relationships both under physiological and diseased states. In defining a metalloproteome, we seek to determine the set of proteins, which have metal-binding capacity, either by virtue of being metalloproteins or having metal-binding sites.

Our research is directed to establishing metalloproteomics, the detailed structural and functional characterization of metal-binding proteins and their structural metal-binding motifs. The establishment of the metalloproteome will provide critically important new information for understanding cellular function physiologically and in disease states arising from metal-associated cytotoxicity. We are developing a hepatic metalloproteome for copper and zinc, using hepatocyte cell lysates for our studies.

The metal-binding proteins are separated by immobilized affinity chromatography (IMAC) and subsequently isolated by ID and 2D gel electrophoresis followed by in-gel digestion. Mass finger printing (MS) and MS/MS measurements on the resulting peptides by both MALDI and ESI QqTOF mass spectrometry are used to determine protein sequences followed by protein database search for identification. Structural characterization of metal-binding proteins are carried out by various spectroscopic techniques including fluorescence, CD, NMR, EPR and XAS.

2. Studies of copper-transporting ATPases:

Copper is an essential element, which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. Although various aspects of copper transport and metabolism have been investigated in the past, very little is known about the specifics of intracellular copper transport. The cloning of the genes responsible for the two major genetic disorders of copper metabolism in humans, Wilson and Menkes diseases, has been a major breakthrough in our understanding of intracellular copper transport.

Both genes are predicted to encode putative copper-transporting P-type ATPases similar to other cation-transporting P-type ATPases. A crucial feature of the copper-transporting ATPases is the presence of a large N-terminal segment, which contains the copper-binding domain. Other features include: eight membrane spanning domains, three intracellular loops and a short C-terminal.

Our studies are directed to a complete characterization of the structure of the N-terminal copper-binding segment by CD, XAS and NMR spectroscopy. In order to study the mechanism of copper-transport we are also investigating the phosphorylation domain for intermolecular interactions and segments of membrane spanning domains for copper interaction. The calcium pump of the sarcoplasmic reticulum, SERCA1a, is a member of the P-type ATPase family whose 2.6 Å resolution crystal structure is available.

In the absence of any crystal structure for Wilson copper-transporting ATPase, we are using comparative modeling to obtain a low-resolution model of the Wilson disease copper-transporting ATPase by homology to the X-ray structure of the calcium pump. The solution structure of the fourth metal-binding domain from the Menkes copper-transporting ATPse is used to model each one of the six heavy metal-associated domains in the N-terminal region. Our experimental results combined with the homology modeling of the copper-transporting ATPase will help to explain the mechanism of copper transport by copper-transporting ATPases.

3. Treatment developments for Menkes and Wilson diseases:

We pioneered the treatment of Menkes disease with copper-histidine. Although patients clearly responded well to the treatment, the severity of the disease in each case remained unresolved. To clarify these questions, we characterized the genetic defects in patients who were treated with copper-histidine. Results revealed severe mutations in these patients indicative of a classic form of Menkes disease. These patients would not have survived without the copper-histidine treatment.

The long-term follow-up (aged ten to 22 years) demonstrated normal or near normal intellectual development in these patients, however, they still showed problems associated with connective tissue abnormalities. To evaluate the effect of copper-histidine on the expression of copper enzymes, the growth of mutant and normal cells in the medium containing copper-histidine is being studied. We plan to use Menkes fibroblasts as well as other cells with missence and splice site mutations. Enzymes being analyzed are those highly relevant to the manifestations of Menkes disease.

Wilson disease is a genetic disorder of copper transport, which causes progressive hepatic or neuropsychiatric diseases in affected individuals. Without treatment, this disease is invariably lethal. Various medical treatments are available, including penicillamine and other oral chelators, but each carries risks of side effects which may be severe or life threatening. A simple, effective, and non-toxic treatment would be highly desirable.

In Menkes disease copper-histidine effectively supplies copper to cells. We hypothesize that the reverse strategy might be effective in Wilson disease: namely to supply excess histidine so that it may remove copper directly from hepatocytes. We anticipate that such treatment would be safer and more effective than existing treatment modalities. We are examining this hypothesis in a mouse model of Wilson disease. Findings from these studies are expected to improve diagnosis and treatment of Wilson disease.

4. Heavy Metals in the Environment:

Our global environment consists of numerous natural and artificial metals. Metals have played a critical role in industrial development and technological advances. Most metals are not destroyed; indeed they are accumulating at an accelerated pace, due to the ever-growing demands of modern society. The widespread distribution of metals in the environment is of great concern because of the toxic properties of many of them. A fine balance must be maintained between metals in the environment and human health.

One of our current research interests is in the area of arsenic in our global environment. It has both carcinogenic and noncarcinogenic potentials. Our interests are in the environmental behaviour of arsenic with special reference to the abundance and distribution of arsenic in soil and drinking water. Studies include human exposure and aspects of human toxicology with special emphasis on chronic arsenic poisoning and its general effects related to dermatological manifestations, cardiovascular diseases, neurological impairments and cancer effects. Plans are underway to focus on a variety of in vitro toxicological screening methods for the biomonitoring of toxic metals.

These methods take advantage of intracellular effects of metals to induce expression of detoxifying proteins, other protective proteins and proteins involved in cell cycle, proliferation and apoptosis. Research is also directed towards the investigation of multi-metal synergy in causing severe arsenic toxicity in certain parts of the world.

Severe chronic arsenic poisoning may be magnified by antimony. Also, chronic arsenic poisoning might be magnified by a lack of selenium, an essential element that prevents toxic effects of arsenic. Arsenic toxicity might also be magnified by a lack of zinc, which is an essential element that promotes the repair of tissue damaged by arsenic. These studies are conducted in a team effort with a group of international scientists probing the arsenic poisoning in our global environment in order to address the health crisis management efforts.

Publications

Frisbie SH, Ortega R, Maynard DM, Sarkar B. The concentrations of Arsenic and other Toxic Elements in Bangladesh's Drinking Water. Environmental Health Perspectives 110, 1147-1153 (2002)

DiDonato M, Hsu M-F, Narindrasorasak S, Que Jr L, Sarkar B. Zinc Binding to the N-Terminal Domain of the Wilson Disease Copper-Transporting ATPase: Implications for in vivo Metal Ion Mediated Regulation of ATPase Activity. J. Biol. Chem. 2002, 277(16), 13409-13414.

Hou Z-J, Narindrasorasak S, Bhushan B, Sarkar B, Mitra B. Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)/Zn(II)/Cd(II)-ATPase from Escherichia coli. J. Biol. Chem. 276, 40858-40863 (2001).

Donaldson LW, Skrynnikov NR, Choy W-Y, Muhandiram DR, Sarkar B, Forman-Kay JD, Kay LE. Structural Characterization of Proteins with an Attached ATCUN Motif by Paramagnetic Relaxation Enhancement NMR Spectroscopy. J Am. Chem. Soc. 123, 9843-9847 (2001).

DiDonato M, Hsu H-F, Narindrasorasak S, Que Jr L, Sarkar B. Copper-Induced Conformational Changes in the N-Terminal Domain of Wilson Disease Copper-Transporting ATPase. Biochemistry 39, 1890-1896 (2000).

Sarkar B. Treatment of Wilson and Menkes Diseases – Medicinal Inorganic Chemistry. Chem. Rev. 99, 2535-2544 (1999).

Christodoulou J, Danks DM, Sarkar B, Baerlocher KE, Casey R, Horn N, Tümer Z, Clarke JTR. Early Treatment of Menkes Disease with Parenteral Copper-Histidine: Longterm Followup of Four Treated Patients. Am. J. Med. Genet. 76: 154-164 (1998).

DiDonato M, Narindrasorasak S, Forbes JR, Cox DW, Sarkar B. Expression, purification and metal binding properties of the N-terminal domain from the Wilson disease putative Cu-transporting ATPase (ATP7B). J. Biol. Chem. 272, 33279-33282 (1997).

Tümer Z, Horn N, Tønnesen T, Christodoulou J, Clarke JTR, Sarkar B. Efficacy of Early Copper-Histidine Treatment for Menkes Disease. Nature Genetics, 12, 11-13 (1996).