Pawel A. Osmulski

Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer

Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial–mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer.

The central unit within the 19S regulatory particle of the proteasome

The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.

Caretaker or undertaker? The role of the proteasome in aging

Despite intensive studies, the molecular basis of the decline of protein degradation with age still remains unresolved. It is suspected that the proteasome is one of the key factors controlling the age-dependent turnover of intracellular proteins. This hypothesis is based on the observation that the proteasome is a part of the ubiquitin-proteasome pathway, which together with the lysosomal pathway constitute the major mechanisms of protein degradation. While there are alterations in proteasome structure and function with age, the observed changes do not provide a clear mechanism for explaining the decline of protein degradation. In addition, there are no consistent changes in the ubiquitination system to account for this decline. On the other hand, because of the essential role played by the proteasome in the maintenance of cellular homeostasis, the observation of age-related changes in structure and function will ultimately be demonstrated to contribute to the aging process. The fact that food restriction, the only currently available experimental paradigm that can alter the aging process, modulates the age-related changes in proteasome structure and function provides presumptive evidence that the proteasome is involved in the aging process.

Altered Composition of Liver Proteasome Assemblies Contributes to Enhanced Proteasome Activity in the Exceptionally Long-Lived Naked Mole-Rat

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life.

A Tetrahedral Transition State at the Active Sites of the 20S Proteasome Is Coupled to Opening of the α-Ring Channel

Intrinsic conformational transitions contribute to the catalytic action of many enzymes. Here we use a single-molecule approach to demonstrate how such transitions are linked to the catalytic sites of the eukaryotic proteasome, an essential protease of the ubiquitin pathway. The active sites of the cylindrical proteasomal core particle are located in a central chamber accessible through gated entry channels. By using atomic force microscopy, we found continual alternation between open and closed gate conformations. We analyzed the relative abundance of these conformers in wild-type and mutated yeast core particles upon exposure to substrates or inhibitors. Our data indicate that the dynamic gate can be opened by allosteric coupling to a tetrahedral transition state at any of the working active centers. The results point to the N(alpha)-amine of the N-terminal active site threonyl residue as the major effector group responsible for triggering the essential conformational switch.

Small-molecule inhibitors of proteasome activity.

The fast-track approval of a proteasome inhibitor, PS-341, to treat multiple myeloma spurred a wave of interest in both the proteasome itself and small-molecule compounds blocking its activities. Besides being candidates for drugs against cancer, autoimmune diseases, inflammation, or stroke, specific proteasome inhibitors are indispensable tools for biochemical and cell biology investigations of the proteasome and proteasome-ubiquitin system. Numerous synthetic peptide derivatives, such as boronates, epoxides, aldehydes, vinyl sulfones, cyclic peptides, and lactones, block the N-terminal threonine-type active centers of the enzyme, halting the cleavage of proteasomal protein substrates both in vitro and in vivo. Because some of the proteasomal inhibitors exhibit a high specificity toward only one particular type of an active center of the proteasome, they constitute valuable probes for testing the mechanism of proteolysis catalyzed by the enzyme. In this chapter we discuss the most common applications of available proteasome inhibitors. In addition to the best-known competitive inhibitors, we also describe the benefits from the use of allosteric inhibitors, which induce distinct but less understood in vitro and in vivo effects on the proteasomal machinery. Finally, we present the application of the basic biochemical procedures to decipher the mechanism of interactions of a novel compound with the proteasome.

Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA

In eukaryotes, the initiation of DNA replication requires the interaction between origin sequences and the origin recognition complex (ORC), which is highly conserved. In this report, atomic force microscopy (AFM) was used to examine the binding of Schizosaccharomyces pombe (sp) ORC and the spOrc4 protein with the sp autonomously replicating sequence 1 (ars1). AFM imaging revealed that spORC binding to ars1 occurred solely through spOrc4p and depended on the N-terminal AT-hook domains present in spOrc4p. At high molar ratios of spORC (or spOrc4p alone) to DNA (6:1), all of the input ars1 was bound in a one protein complex to one plasmid manner. Restriction digestion and AFM analysis of protein–DNA fragments revealed the presence of two binding sites in ars1. One site mapped to a region centered at nucleotide 838 of ars1 previously detected by DNase I protection that was reported to be essential for the autonomously replicating sequence activity of ars1. The second site mapped to a previously uncharacterized region centered at nucleotide 1148. AFM showed that the length of the DNA fragment complexed with either spORC or spOrc4p was shortened by ≈140 bp, suggesting the wrapping of two turns of the DNA around the spOrc4p alone as well as the spOrc4p in spORC. We also show that treatment of the spORC (spOrc4p)–ars1 complex with topoisomerase I induced a negative shift in the topoisomer distribution. These findings suggest that the binding of spORC to origin DNA alters the structure of the DNA. Thus, in the case of spORC, due to its unusual spOrc4p, at least two factors are likely to influence ars1 activation. These include the selective binding of the complex to A- and T-rich regions and the alteration of the DNA structure due to its wrapping around spOrc4p.

Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer’s disease

Amyloid beta (Aβ) is implicated in Alzheimers disease (AD) as an integral component of both neural toxicity and plaque formation. Brains of the longest-lived rodents, naked mole-rats (NMRs) approximately 32 years of age, had levels of Aβ similar to those of the 3xTg-AD mouse model of AD. Interestingly, there was no evidence of extracellular plaques, nor was there an age-related increase in Aβ levels in the individuals examined (2-20+ years). The NMR Aβ peptide showed greater homology to the human sequence than to the mouse sequence, differing by only 1 amino acid from the former. This subtle difference led to interspecies differences in aggregation propensity but not neurotoxicity; NMR Aβ was less prone to aggregation than human Aβ. Nevertheless, both NMR and human Aβ were equally toxic to mouse hippocampal neurons, suggesting that Aβ neurotoxicity and aggregation properties were not coupled. Understanding how NMRs acquire and tolerate high levels of Aβ with no plaque formation could provide useful insights into AD, and may elucidate protective mechanisms that delay AD progression.

The Proteasome in Health and Disease

The giant proteolytic factory called the proteasome came a long way from a biochemical curio to a major regulator of cellular physiology and a renowned drug target within the ubiquitin proteasome pathway (UPP). Thanks to availability of highly specific inhibitors of the proteasome, in less than twenty years it was possible to identify major transcription factors, cyclins, and products of oncogenes as crucial substrates for the UPP. Nine years passed since the FDA speedily approved bortezomib, the inhibitor of proteasome, for treatment of multiple myeloma. One year after its approval, the field was honored by awarding the Nobel Prize to Hershko, Ciechanover and Rose for introducing the concept of controlled proteolysis of ubiquitin-tagged substrates, with proteasome as the intracellular recycling facility. Taking into consideration the universal involvement of the proteasome in the life of all cells in human body, it comes to no surprise that the enzyme is deeply implicated in etiology, progression, diagnosis or cure of multiple diseases. Below we discuss some aspects of the involvement: from direct causative links to changes in proteasome properties that correlate with pathological conditions. We start with diseases collectively known as cancer, and with immune system-related pathologies. Here, the proteasome inhibitors are either already used in clinics, or undergo advanced preclinical screening. Then, we will continue with cardiovascular disorders, followed by aging. Changes of the proteasome make-up during aging may be a priming factor for neurodegenerative diseases, described last. We discuss the potential for proteasome regulation: inhibition, activation or specificity modulation, to successfully enter the clinical setting.

The Growth-Suppressive Function of the Polycomb Group Protein Polyhomeotic Is Mediated by Polymerization of Its Sterile Alpha Motif (SAM) Domain

Background: The mechanism by which Sterile Alpha Motifs (SAMs) self-associate and polymerize to control protein function is unknown. Results: SAM polymerization in Polyhomeotic, a Polycomb group protein, is controlled by an unstructured linker sequence in Polyhomeotic. Conclusion: Polyhomeotic growth suppressive function is enhanced by increasing SAM polymerization. Significance: Functions of other SAM domain-containing proteins could be manipulated through their unstructured linkers. Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.