A central component of a number of degenerative diseases is the deposition of protein as amyloid fibers. Self-assembly of amyloid occurs by a nucleation-dependent mechanism that gives rise to a characteristic sigmoidal reaction profile. The abruptness of this transition is a variable characteristic of different proteins with implications to both chemical mechanism and the aggressiveness of disease. Because nucleation is defined as the rate-limiting step, we have sought to determine the nature of this step for a model system derived from islet amyloid polypeptide. We show that nucleation occurs by two pathways: a fiber-independent (primary) pathway and a fiber-dependent (secondary) pathway. We first show that the balance between primary and secondary contributions can be manipulated by an external interface. Specifically, in the presence of this interface, the primary mechanism dominates, whereas in its absence, the secondary mechanism dominates. Intriguingly, we determine that both the reaction order and the enthalpy of activation of the two nucleation processes are identical. We interrogate this coincidence by global analysis using a simplified model generally applicable to protein polymerization. A physically reasonable set of parameters can be found to satisfy the coincidence. We conclude that primary and secondary nucleation need not represent different processes for amyloid formation. Rather, they are alternative manifestations of the same, surfacecatalyzed nucleation event.amylin ͉ fibers ͉ islet amyloid polypeptide ͉ nucleation T he noncovalent, fibrous self-assembly of proteins, including hemoglobin S, actin, microtubules, and amyloid fibers, occurs by a nucleation-dependent mechanism (1-5). If polymer product formation is monitored as a function of time in a reaction where all of the protein is initially monodisperse, there is a lag phase in which little product is formed, followed by a period of rapid growth (2). Such observations are consistent with a rate-limiting step in which change occurs to a high-energy intermediate known as the nucleus. In many systems such as actin, collagen, and microtubules, the nucleus is modeled as an oligomeric species that is in a highly unfavorable equilibrium with monomer (1, 3-6). The nucleus may also be a high-energy conformation of monomer, such as that reported for polyglutamine (7). Regardless, the rate-limiting step involves a nucleus because it is defined as the species with the highest free energy and therefore lowest population (1, 4).This model of nucleation alone cannot account for the high apparent cooperativity of conversion observed in many systems. Historically, hemoglobin S was the first biological polymerization reaction to demonstrate a transition time that is much shorter than its preceding lag phase (8). This same phenomenon is commonly reported for amyloid systems including islet amyloid polypeptide (IAPP) from type II diabetes (9), A␤ from Alzheimer's (10), PrP from the mammalian prion (11), and Sup35 from the yeast prion (12, 13), as well as mode...

The proteasome is an intracellular enzyme complex that degrades ubiquitin-tagged proteins and thereby regulates protein levels within the cell. Given this important role in maintaining cellular homeostasis, it is perhaps somewhat surprising that proteasome inhibitors have a therapeutic window. Proteasome inhibitors have demonstrated clinical efficacy in the treatment of multiple myeloma and mantle cell lymphoma and are under evaluation for the treatment of other malignancies. Bortezomib is the first and only Food and Drug Administration-approved proteasome inhibitor that inhibits this enzyme complex in a reversible fashion. Although bortezomib improves clinical outcomes when used as a single agent, most patients do not respond to this drug and those who do respond almost uniformly relapse. As such, efforts are underway to develop proteasome inhibitors that act through mechanisms distinct from that of bortezomib. Specifically, inhibitors that bind the active site of the proteasome and inhibit the complex irreversibly have been developed and are in advanced clinical trials. Inhibitors that act on sites of the proteasome outside of the catalytic center have also been identified and are in preclinical development. In this review, we discuss the structure and function of the proteasome. We then focus on the molecular biology, chemistry, and the preclinical and clinical efficacy of novel proteasome inhibitors as strategies to inhibit this target and overcome some forms of bortezomib resistance.

The development of new protein labeling strategies, along with optimized experiments that exploit the label, have significantly impacted on the types of biochemical problems that can now be addressed by solution NMR spectroscopy. Here we describe how methyl labeling of key residues in a highly deuterated protein background has facilitated studies of the structure, dynamics and interactions of supra-molecular particles. The methyl-labeling approach is briefly reviewed, followed by a summary of applications to three different molecular machines so as to illustrate the types of questions that can now be addressed. Areas where future innovations will lead to yet further improvements are highlighted as well.