Androgen deprivation-induced elevated nuclear SIRT1 promotes prostate tumor cell survival by reactivation of AR signaling.

The NAD+-dependent deacetylase, Sirtuin 1 (SIRT1) is involved in prostate cancer pathogenesis. However, the actual contribution is unclear as some reports propose a protective role while others suggest it is harmful. We provide evidence for a contextual role for SIRT1 in prostate cancer. Our data show that (i) mice orthotopically implanted with SIRT1-silenced LNCaP cells produced smaller tumors; (ii) SIRT1 suppression mimicked AR inhibitory effects in hormone responsive LNCaP cells; and (iii) caused significant reduction in gene signatures associated with E2F and MYC targets in AR-null PC-3 and E2F and mTORC1 signaling in castrate-resistant ARv7 positive 22Rv1 cells. Our findings further show increased nuclear SIRT1 (nSIRT1) protein under androgen-depleted relative to androgen-replete conditions in prostate cancer cell lines. Silencing SIRT1 resulted in decreased recruitment of AR to PSA enhancer selectively under androgen-deprivation conditions. Prostate cancer outcome data show that patients with higher levels of nSIRT1 progress to advanced disease relative to patients with low nSIRT1 levels. Collectively, we demonstrate that lowering SIRT1 levels potentially provides new avenues to effectively prevent prostate cancer recurrence.

Potential allosteric modulators of the proteasome activity.

Proteasome, consisting of a tube-shaped proteolytic core particle and attached to it regulatory modules, is a multifunctional enzymatic complex essential for the ubiquitin-proteasome metabolic pathway. Due to its immense involvement in regulation of cellular physiology, the proteasome is an acknowledged anticancer drug target and potential target to treat inflammatory or degenerative diseases. So far, competitive inhibitors of the core particle gain most consideration as drugs. We postulate that noncompetitively-acting small-molecule compounds would provide excellent means to precisely regulate actions of the proteasome. In this study, we evaluated five short peptides based on sequences of two proteins known to interact with the core proteasome: HIV-1 Tat and PA28/REG activator. We performed Circular Dichroism (CD), Fourier Transformed Infrared Spectroscopy (FTIR), and Nuclear Magnetic Resonance (NMR) analysis, supplemented by MD simulations, and tested influence of the peptides on performance of the core particle active sites and functioning of regulatory modules. We found that PP2-containing Tat peptides are noncompetitive inhibitors of the core, interfering with the actions of PA28alphabeta activator. In addition, at low concentrations the turn-prone Tat2 is able to activate the latent core. The random coil-structured PA28-derived peptides display only weak or nondetectable direct effects on the core activities, exhibiting, however, a positive cooperation with activity-enhancing actions of PA28alphabeta.

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.

Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast.

The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells. The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings. The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role. Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed. Because of this result, the path of substrates into the catalytic chamber has remained enigmatic. We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast. We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel. The preferred conformation depends on the ligands present. Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation. These results shed new light on the possible path of the substrate into the proteolytic chamber.

A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast.

One eukaryotic proteolytic complex--the proteasome--is classed as the major nonlysosomal protease, by its known and suspected functions, its size and its complexity. It seems improbable that other enzymes may be capable of substituting, even partially, for the potent proteasome, as this complex has a vital role in many cellular processes. Nevertheless, it is possible to adapt cultured EL-4 mouse lymphoma cells to survive in the presence of a specific inhibitor of the proteasome. The inhibition of the proteasome in these adapted EL-4 cells is accompanied by a dramatic increase in the activity of a new, as yet uncharacterized, large proteolytic complex. Here, we have presented evidence that a similar proteolytic activity is constitutively present in fission yeast, Schizosaccharomyces pombe, and that the yeast and mouse enzymes share basic physicochemical properties. We have shown that the S. pombe protease is found in two stable oligomeric forms, both of which are peptidases, although only the larger form acts as a proteinase. The relative amounts of the large and the small forms of the protease in the complex depended on the growth phase of the yeast culture and affected enzyme activity, suggesting that the activity of the enzyme is regulated by its oligomerization status. We refer to the new proteolytic complex as the 'multicorn' to indicate its analogy to the archaebacterial tricorn protease.

High-pressure-assisted reconstitution of recombinant chloroperoxidase.

An expression vector containing a T7 promoter and an OmpA signal sequence followed by the DNA sequence of mature chloroperoxidase from the fungus Caldariomyces fumago has been transformed into Escherichia coli. This construct gave high-level expression of apochloroperoxidase when induced with isopropyl thiogalactopyranoside. The nonglycosylated apoenzyme was secreted into periplasmic space. The recombinant apochloroperoxidase was expressed at a level representing about 2% of the total cellular protein. Before conversion to holoenzyme, the apochloroperoxidase was denatured in 8 M urea and partially purified by DEAE chromatography. Maximum yields of holoenzyme were obtained when the denatured apochloroperoxidase, dissolved in a refolding buffer containing iron protoporphyrin IX, calcium ions, and oxidized glutathione, was subjected to high pressure (207 MPa) at -12 degrees C and then allowed to refold at atmospheric pressure and room temperature. The recombinant holoenzyme was characterized by absorption and CD spectroscopy and tested for halogenation and peroxidation activity. The yield of active holochloroperoxidase was about 5% when high-pressure treatment was used as part of the reconstitution process. In the absence of pressure treatment, holoenzyme was formed at about the 1% level. The holochloroperoxidase preparations which resulted from high-pressure treatment showed, upon return to atmospheric pressure, a considerably higher content of native-like secondary structure compared to the nonpressurized preparations. These experiments show that active recombinant chloroperoxidase molecules can be produced, and prove that glycosylation is not a mandatory requirement for chloroperoxidase refolding.

Spectroscopic studies of an insect hemoglobin from the backswimmer Buenoa margaritacea (Hemiptera:Notonectidae).

Hemoglobin (Hb) isolated from the backswimmer Buenoa margaritacea has been analyzed spectroscopically. The met form at pH less than 6 shows a 30nm red shift in the Qv and Qo bands and a 5nm red shift in the Soret band compared to mammalian Hb, while only minor differences are seen in the spectra of the CO and O2 adducts of Hb from Buenoa and mammals. EPR spectra of the metHb show a superposition of signals; at low pH they are mainly of axial high-spin character, while at high pH a low-spin signal predominates with an O-type g-tensor (2.54, 2.61, 1.85) comparable to that of hydroxy myoglobin. Infrared spectra of Hb12C-16O at pH 8.2 reveal two major absorption bands at 1934 cm-1 and 1967 cm-1, which shift to 1892 cm-1 and 1923 cm-1, respectively, for Hb12C-18O. As isolated the Buenoa Hb consists of several isozymes, all of which have a histidine as the proximal ligand of the heme iron.

Radiation-induced changes of structural and functional properties of human hemoglobin. I. Spectral characterization of irradiated deoxyhemoglobin.

Structural changes of human deoxyhemoglobin (deoxyHb) induced by water and ethanol radicals were investigated in this study using absorption spectroscopy. Spectra of deoxyHb samples irradiated under various conditions (the atmosphere of argon or N2O in the absence or presence of ethanol) indicate their conversion into methemoglobin (metHb), hemichrome- and cholehemichrome-like products. The absorbance at the characteristic maxima of these derivatives and also of the oxidized or reduced samples following irradiation depends on the dose of radiation and the conditions employed.

Radiation-induced changes of structural and functional properties of human hemoglobin. II. Structural and functional characterization of irradiated deoxyhemoglobin.

Gel filtration and SDS-PAGE separation of hemoglobin (Hb) irradiated under argon or N2O show formation of covalent-aggregated Hb molecules. The production of covalent bonds is attributed mainly to the action of hydroxyl radicals, because addition of ethanol, a scavenger of these radicals, suppresses this reaction to a great extent. The oxidized heme iron forming metHb or hemichromes is found in all the separated fractions of irradiated Hb. It is also found that the radiation-modified Hb molecules exhibit a decrease of co-operative binding of oxygen.