Biomedical subjects

Pawel A Osmulski

Publications and source records attributed to Pawel A Osmulski.

9 recordsLinked to original sources

PubMed2006

Highbrow proteasome in high-throughput technology.

Proteasome is a major protease of the ubiquitin-proteasome pathway involved in the regulation of practically all intracellular biochemical processes. The enzyme core is created by a heteromultimer of complex architecture built with multiple subunits arranged into a tube-like structure. The multiple active sites of diverse peptidase specificity are hidden inside the tube. Access to the interior is guarded by a gate formed by the N-termini of specialized subunits and by the attachment of additional multisubunit protein complexes controlling the enzymatic capabilities of the core. Proteasome, due to its Byzantine molecular architecture and equally sophisticated enzymatic mechanism, is by itself a fascinating biophysical object. Recently, the position of the protease advanced from an academically remarkable protein processor to a providential anticancer drug target and futuristic nanomachine. Proteomics studies actively shape our current understanding of the protease and direct the future applications of the proteasome in medicine.

Allosteric Regulation↗

PubMed2006

Allosteric regulators of the proteasome: potential drugs and a novel approach for drug design.

The proteasome recently gained an exceptional attention as a novel drug target, therefore its inhibitors became important subjects for rational drug design. A synthetic competitive inhibitor Velcade was lately approved in a fast-track process to treat multiple myeloma and is tested with other types of cancers. The proteasome is a major proteolytic assembly in eukaryotic cells responsible for the degradation of most intracellular proteins, including proteins crucial to cell cycle regulation and apoptosis. The ubiquitin-proteasome pathway has been implicated in many diseases such as cancer, autoimmune diseases, inflammation, and stroke. The activity of the proteasome can be blocked for therapeutic purposes with competitive inhibitors like Velcade, which trigger apoptosis in target cells. However, much more versatile outcomes and a true control of the proteasome can be achieved with allosteric regulators. Certain natural proteins and peptides bind to the catalytic core of the proteasome and allosterically induce a wide array of effects ranging from changes in product size to substrate-specific inhibition. Designing small synthetic compounds allosterically interacting with the proteasome represents a novel approach that has enormous potential for the treatment of a wide range of diseases. Below we provide a review of current knowledge about proteasomal allosteric ligands.

Allosteric Regulation↗

PubMed2005

Atomic force microscopy of the proteasome.

The proteasome should be an ideal molecule for studies on large enzymatic complexes, given its multisubunit and modular structure, compartmentalized design, numerous activities, and its own means of regulation. Considering the recent increased interest in the ubiquitin-proteasome pathway, it is surprising that biophysical approaches to study this enzymatic assembly are applied with limited frequency. Methods including atomic force microscopy, fluorescence spectroscopy, surface plasmon resonance, and high-pressure procedures all have gained popularity in characterization of the proteasome. These methods provide significant and often unexpected insight regarding the structure and function of the enzyme. This chapter describes the use of atomic force microscopy for dynamic structural studies of the proteasome.

Animals↗

PubMed2005

Characterization of noncompetitive regulators of proteasome activity.

The success of bortezomib, a competitive proteasome inhibitor and a drug approved to treat multiple myeloma, spurred interest in compounds targeting catalytic sites of the enzyme. The aim of this chapter, however, is to focus attention on the small molecule, natural or synthetic compounds binding far away from the catalytic centers, yet modifying the performance of the proteasome. Defining allostery broadly as any kind of ligand-induced, long-distance transfer of conformational signals within a molecule, most such compounds are allosteric effectors capable of regulating the proteasome in vitro and in vivo in a manner more diverse and precise than competitive inhibitors. Proline- and arginine-rich peptides (PR peptides) are examples of such compounds and are currently being considered as potential drugs with anti-inflammatory and proangiogenic activities. This chapter describes a set of methods useful for characterizing the effects of such inhibitors on the proteasome.

Allosteric Regulation↗

PubMed2005

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.

Animals↗

PubMed2004

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 approximately 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.

DNA↗

PubMed2004

DNA-looping by RXR tetramers permits transcriptional regulation "at a distance".

RXR, a member of the superfamily of nuclear hormone receptors, regulates gene transcription in response to 9-cis-retinoic acid. We previously showed that, among nuclear receptors, RXR is unique in that it self-associates into homotetramers, and that these tetramers dissociate rapidly upon ligation. Here, we report that binding of RXR tetramers to DNA containing two RXR response elements results in a dramatic DNA-looping. RXR can thus juxtapose distant DNA sequences, enabling transcriptional regulation by far-upstream factors. We show that RXR functions as a DNA architectural factor and that, while this activity is regulated by 9-cis-retinoic acid, it is distinct from and independent of the receptor's intrinsic transcriptional activity. The data establish RXR as the first identified architectural factor whose activity is regulated by a small ligand, and demonstrate a novel mechanism of transcriptional regulation by retinoids.

Animals↗

PubMed2003

Proline- and arginine-rich peptides constitute a novel class of allosteric inhibitors of proteasome activity.

Substrate-specific inhibition of the proteasome has been unachievable despite great interest in proteasome inhibitors as drugs. Recent studies demonstrated that PR39, a natural proline- and arginine-rich antibacterial peptide, stimulates angiogenesis and inhibits inflammatory responses by specifically blocking degradation of IkappaBalpha and HIF-1alpha by the proteasome. However, molecular events involved in the PR39-proteasome interaction have not been elucidated. Here we show that PR39 is a noncompetitive and reversible inhibitor of the proteasome function. This effect is achieved by a unique allosteric mechanism allowing for specific inhibition of degradation of selected proteins without affecting total proteasome-dependent proteolysis. Atomic force microscopy (AFM) studies demonstrate that 20S and 26S proteasomes treated with PR39 or its derivatives exhibit serious perturbations in their structure and their normal allosteric movements. These effects are universal for proteasomes from yeast to human. The shortest functional sequence derived from PR39 still showing the allosteric inhibitory effect consists of eleven NH(2)-terminal residues containing essential three NH(2)-terminal arginines. The noncompetitive and reversible in vitro action of PR39 and its truncated derivatives is matched by the ability of the peptides to induce angiogenesis in vivo. We postulate that PR39 changes conformational dynamics of the proteasomes by interactions with the noncatalytic subunit alpha7 in a way that prevents the enzyme from cleaving the substrates of unique structural constraints.

Allosteric Site↗

PubMed2002

Nanoenzymology of the 20S proteasome: proteasomal actions are controlled by the allosteric transition.

The proteasome is a major cytosolic proteolytic assembly, essential for the physiology of eukaryotic cells. Both the architecture and enzymatic properties of the 20S proteasome are relatively well understood. However, despite longstanding interest, the integration of structural and functional properties of the proteasome into a coherent model explaining the mechanism of its enzymatic actions has been difficult. Recently, we used tapping mode atomic force microscopy (AFM) in liquid to demonstrate that the alpha-rings of the proteasome imaged in a top-view position repeatedly switched between their open and closed conformations, apparently to control access to the central channel. Here, we show with AFM that the molecules in a side-view position acquired two stable conformations. The overall shapes of the 20S particles were classified as either barrel-like or cylinder-like. The relative abundance of the two conformers depended on the nature of their interactions with ligands. Similarly to the closed molecules in top view, the barrels predominated in control or inhibited molecules. The cylinders and open molecules prevailed when the proteasome was observed in the presence of peptide substrates. Based on these data, we developed the two-state model of allosteric transitions to explain the dynamics of proteasomal structure. This model helps to better understand the observed properties of the 20S molecule, and sets foundations for further studies of the structural dynamics of the proteasome.

Allosteric Regulation↗