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Michael Maurizi, Ph.D.

Portait Photo of Michael Maurizi
Laboratory of Cell Biology
Head, Biochemistry of Proteins Section
Senior Investigator
Center for Cancer Research
National Cancer Institute
Building 37, Room 2128B
Bethesda, MD 20892-4256
Phone:  
301-496-7961
Fax:  
301-402-0450
E-Mail:  
mmaurizi@helix.nih.gov

Biography

Dr. Maurizi received his Ph.D. in biochemistry from the University of Illinois and completed postdoctoral studies with Earl Stadtman and Ann Ginsburg in the National Heart, Lung, and Blood Institute at the NIH. Dr. Maurizi began his work on energy-dependent proteases in collaboration with Susan Gottesman in the Laboratory of Molecular Biology, NCI, before joining the staff of the Laboratory of Cell Biology in 1991.

Research

Structure and Function of ATP-Dependent Proteases Involved in Rapid Intracellular Protein Degradation in Prokaryotes and Eukaryotes

Intracellular protein degradation is a major post-translational regulatory mechanism that comes into play in virtually every aspect of the biology of a cell. Crucial roles for protein degradation include timing of the cell division cycle, heat shock and other stress responses, adaptation to changes in nutrient availability, cell signaling mechanisms and developmental changes, response to DNA damage, viral infections, and cell death pathways. Protein degradation also serves a vital function in protein quality control, removing mistranslated, damaged, denatured, and otherwise conformationally abnormal proteins from cells. Protein quality control by proteases is accomplished in partnership with molecular chaperones. Together, the chaperones and proteases constitute a triage system that recognizes abnormal proteins and shuttles them into repair or destruction pathways.
Most intracellular proteolysis is performed by ATP-dependent proteases, which unite a chaperone and a protease activity in a single enzyme complex. Our studies have focused on the ATP-dependent proteases from E. coli and their close homologs from mammalian mitochondria. Our studies have revealed remarkable parallels in composition and structure between the E. coli ATP-dependent proteases and those in higher organisms, including the 26S proteasome, the major ATP-dependent protease in the cytosol of eukaryotic cells. These parallels underlie a set of mechanistic principles that is common to all ATP-dependent proteases. ATP-dependent proteases are high molecular weight, multimeric proteins containing at least two types of enzymatic functions, an ATP-dependent protein unfoldase, referred to as the chaperone component, and an endopeptidase, referred to as the protease component. In addition, the complexes contain one or more auxiliary components, either as intrinsic domains or as components that associate with the complex or its substrates. The chaperone and protease functions are contributed by separate domains, which may be connected tandemly in a single polypeptide, as in the E. coli proteases, Lon and FtsH, or non-covalently assembled from different polypeptides, as in the Clp proteases and the proteasomes. The protease domains are arranged so that the proteolytic active sites are sequestered within the oligomeric assembly and are inaccessible to folded polypeptides and proteins. In a reaction cycle, protein substrates bind to the chaperone component, undergo some degree of unfolding, and are either released and allowed to refold or are translocated into the protease component and degraded. Thus, substrates are kinetically partitioned between degradation and release depending either on intrinsic properties of the substrate or on extrinsic factors that favor one or the other pathway. Binding and release of substrates are controlled by cycles of ATP binding and hydrolysis. Additional regulatory components, adaptor proteins, affect substrate binding or retention and thus influence coupling between changes in nucleotide state and binding or release of substrates. The mechanistic details of these processes are being investigated.
Crystal structure analysis of ClpA and the chaperone domains of other ATP-dependent proteases have shown that their nucleotide binding module belong to the AAA+ superfamily (ATPases associated with various cellular activities). AAA+ proteins are molecular machines involved in conformational reconfiguration of macromolecular complexes. The AAA+ module is designed to couple ATP hydrolysis to conformational changes in the chaperone, which then imparts a mechanical force to portions of a bound substrate. This force can be used to transiently disrupt internal interactions within the substrate, allowing the substrate to undergo structural rearrangement, promoting remodeling of complexes involving the substrate, or permitting the substrate to translocate along or through another macromolecule. For ATP-dependent proteolysis to occur, both the structural disruption and translocation activities are required, because the proteolytic sites lie within an internal chamber formed by association of the protease subunits. In the ClpP protease, two rings of seven subunits interact to enclose an active site chamber accessible only through narrow channels along the seven-fold symmetry axes. ClpP associates with two related chaperones, ClpA, which has two ATPase domains, and ClpX, which has only one. ClpA and ClpX form six-membered rings and stack on both faces of the double ClpP ring. The complexes are hollow cylinder with the degradation chamber in the center. ClpAP and ClpXP complexes remain intact during multiple proteolytic cycles. One cycle, which includes substrate binding, unfolding and translocation, and complete degradation, occurs in 5-10 sec for a 30 kDa substrate protein.
Substrate recognition sites are located within the chaperone component, and the mechanism of interaction and the nature and location of the binding sites are not known. Substrate interaction occurs in several stages: initial binding interactions that bring the substrate and the chaperone together, a committed binding step that imposes some barrier to reversal, and subsequent binding interactions that occur while the substrate is in transit to the protease. Electron microscopy has shown that substrates initially interact at the ends of complexes, indicating that at least some binding sites are located on the ring surface of the chaperone. Whether these are the sites that confer specificity for degradation is not clear. Most motifs that serve as degradation tags for substrates are located at either the N-terminus or C-terminus of the substrate. Such terminally located motifs might also have access to sites within the axial channel, on the peripheral sides of the rings, or even between the rings or between subdomains of the chaperone. Alternatively, specific binding might occur only after higher affinity sites become available due to conformational changes within the chaperone. Our laboratory is working to obtain additional high-resolution structural data to go with biochemical and mutagenesis studies aimed at a better understanding of how substrates interact with the chaperones and how bound proteins are unfolded and translocated into the proteolytic chambers of the complex.


Collaborators on this research include Di Xia, LCB/NIH; Alex Wlodawer, NCI, Frederick; Ann Ginsburg and Greg P, LB, NHLBI; Susan Gottesman, LMB, NCI; Alasdair Steven, LSBR, NIAMS; Bijan Ahvazi, LSB, NIAMS.

This page was last updated on 6/7/2013.