Maria Gabriella Santoro

Activation of the Heat Shock Factor 1 by Serine Protease Inhibitors AN EFFECT ASSOCIATED WITH NUCLEAR FACTOR-κB INHIBITION

Heat shock proteins (HSPs) have a cytoprotective role in several human diseases, including ischemia and viral infection. Nuclear factor-κB (NF-κB) is a critical regulator of inflammation and virus replication. Here we report that a class of serine protease inhibitors with NF-κB-inhibitory activity are potent HSP inducers via activation of heat shock transcription factor 1 (HSF1) in human cells. 3,4-Dichloroisocoumarin, the most effective compound, rapidly induces HSF1 DNA binding activity and phosphorylation, leading to transcription and translation of heat shock genes for a period of several hours. HSF1 activation is independent of de novo protein synthesis and is correlated in a concentration- and time-dependent manner with NF-κB inhibition. Cysteine protease inhibitors E64 and calpain inhibitor II, which do not block NF-κB activation, do not induce HSF DNA binding activity. HSP induction by 3,4-dichloroisocoumarin is associated with antiviral activity during rhabdovirus infection. These results identify a new class of HSP inducers and indicate a link between the regulatory pathways of HSF and NF-κB, suggesting novel strategies to simultaneously switch on cytoprotective genes and down-regulate inflammatory and viral genes.

Novel Human Immunodeficiency Virus Type 1 Protease Mutations Potentially Involved in Resistance to Protease Inhibitors

ABSTRACT Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naïve patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 Å of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naïve patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.

High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations

ABSTRACT To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.

Induction of apoptosis in estrogen receptor-negative breast cancer cells by natural and synthetic cyclopentenones: role of the IkappaB kinase/nuclear factor-kappaB pathway

Nuclear factor-κB (NF-κB), a transcription factor with a critical role in promoting inflammation and cell survival, is constitutively activated in estrogen-receptor (ER)-negative breast cancer and is considered a potential therapeutic target for this type of neoplasia. We have previously demonstrated that cyclopentenone prostaglandins are potent inhibitors of NF-κB activation by inflammatory cytokines, mitogens, and viral infection, via direct binding and modification of the β subunit of the IκB kinase complex (IKK). Herein, we describe the NF-κB-dependent anticancer activity of natural and synthetic cyclopentenone IKK inhibitors. We demonstrate that the natural cyclopentenone 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) is a potent inhibitor of constitutive IκB-kinase and NF-κB activities in chemotherapy-resistant ER-negative breast cancer cells. 15d-PGJ2-induced inhibition of NF-κB function is rapidly followed by down-regulation of NF-κB-dependent antiapoptotic proteins cIAPs 1/2, Bcl-XL, and cellular FLICE-inhibitory protein, leading to caspase activation and induction of apoptosis in breast cancer cells resistant to treatment with paclitaxel and doxorubicin. We then demonstrate that the cyclopentenone ring structure is responsible for these activities, and we identify a new synthetic cyclopentenone derivative, 3-tert-butyldimethylsilyloxy-5-(E)-iso-propylmethylenecyclopent-2-enone (CTC-35), as a potent NF-κB inhibitor with proapoptotic activity in ER-negative breast cancer cells. The results open new perspectives in the search for novel proapoptotic molecules effective in the treatment of cancers presenting aberrant NF-κB regulation.

Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation

ABSTRACT Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC50s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.

2-Cyclopenten-1-one and prostaglandin J2 reduce restenosis after balloon angioplasty in rats: role of NF-κB

The aim of this study was to evaluate, using a rat model of balloon angioplasty, whether prostaglandin (PG) J2 and 2‐cyclopenten‐1‐one are able to reduce restenosis. We found that both PGJ2 and 2‐cyclopenten‐1‐one, administered by local application on carotid arteries, caused a dose‐dependent inhibition of neointimal formation. Furthermore, both agents prevented vascular negative remodeling. The effect of these compounds on restenosis was correlated with an inhibition of nuclear factor‐κB (NF‐κB) activation as well as of intercellular adhesion molecule‐1 (ICAM‐1) protein expression in injured carotid arteries of control animals. Our results show that cyclopentenone PGs and their derivatives reduce restenosis and may have therapeutic relevance for the prevention of human restenosis.

The Proteasome Inhibitor Bortezomib Is a Potent Inducer of Zinc Finger AN1-type Domain 2a Gene Expression: ROLE OF HEAT SHOCK FACTOR 1 (HSF1)-HEAT SHOCK FACTOR 2 (HSF2) HETEROCOMPLEXES*

Background: AIRAP is a recently identified heat shock protein whose function and regulation are still not well defined. Results: Proteasome inhibition causes abundant AIRAP expression in human primary cells. Conclusion: The anticancer drug bortezomib regulates AIRAP expression through HSF1-HSF2 interplay. Significance: This study reveals how persistent proteasomal inhibition by clinically relevant concentrations of bortezomib affects heat shock response regulation and HSF-mediated AIRAP induction. The zinc finger AN1-type domain 2a gene, also known as arsenite-inducible RNA-associated protein (AIRAP), was recently identified as a novel human canonical heat shock gene strictly controlled by heat shock factor (HSF) 1. Little is known about AIRAP gene regulation in human cells. Here we report that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, is a potent inducer of AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated protein accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of HSF2, a factor implicated in differentiation and development regulation. Different from heat-mediated induction, in bortezomib-treated cells, HSF1 and HSF2 interact directly, forming HSF1-HSF2 heterotrimeric complexes recruited to a specific heat shock element in the AIRAP promoter. Interestingly, whereas HSF1 has been confirmed to be critical for AIRAP gene transcription, HSF2 was found to negatively regulate AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor under stress conditions. AIRAP function is still not defined. However, the fact that AIRAP is expressed abundantly in primary human cells at bortezomib concentrations comparable with plasma levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment.

Heat stress triggers apoptosis by impairing NF-kappaB survival signaling in malignant B cells.

Nuclear factor-κB (NF-κB) is involved in multiple aspects of oncogenesis and controls cancer cell survival by promoting anti-apoptotic gene expression. The constitutive activation of NF-κB in several types of cancers, including hematological malignancies, has been implicated in the resistance to chemo- and radiation therapy. We have previously reported that cytokine- or virus-induced NF-κB activation is inhibited by chemical and physical inducers of the heat shock response (HSR). In this study we show that heat stress inhibits constitutive NF-κB DNA-binding activity in different types of B-cell malignancies, including multiple myeloma, activated B-cell-like (ABC) type of diffuse large B-cell lymphoma (DLBCL) and Burkitts lymphoma presenting aberrant NF-κB regulation. Heat-induced NF-κB inhibition leads to rapid downregulation of the anti-apoptotic protein cellular inhibitor-of-apoptosis protein 2 (cIAP-2), followed by activation of caspase-3 and cleavage of the caspase-3 substrate poly(adenosine diphosphate ribose)polymerase (PARP), causing massive apoptosis under conditions that do not affect viability in cells not presenting NF-κB aberrations. NF-κB inhibition by the proteasome inhibitor bortezomib and by short-hairpin RNA (shRNA) interference results in increased sensitivity of HS-Sultan B-cell lymphoma to hyperthermic stress. Altogether, the results indicate that aggressive B-cell malignancies presenting constitutive NF-κB activity are sensitive to heat-induced apoptosis, and suggest that aberrant NF-κB regulation may be a marker of heat stress sensitivity in cancer cells.

Human NF-κB repressing factor acts as a stress-regulated switch for ribosomal RNA processing and nucleolar homeostasis surveillance

Significance Quality control of ribosomal RNA (rRNA) processing is critical for ribosome biogenesis, nucleolar homeostasis, and cell survival; however, the molecular mechanisms governing rRNA processing under stress conditions are poorly understood. This study identifies human NF-κB repressing factor (NKRF) as a HSF1 target gene essential for nucleolar homeostasis during proteotoxic stress. Rather than preventing protein misfolding and/or aggregation, this unconventional stress protein has a critical role in preventing aberrant rRNA precursors and discarded fragment accumulation and directing rRNA processing dynamics. The findings highlight a key aspect of the human cell response to proteotoxic stress, opening new scenarios on ribosome biogenesis regulation. The nucleolus, a dynamic nuclear compartment long regarded as the cell ribosome factory, is emerging as an important player in the regulation of cell survival and recovery from stress. In larger eukaryotes, the stress-induced transcriptional response is mediated by a family of heat-shock transcription factors. Among these, HSF1, considered the master regulator of stress-induced transcriptional responses, controls the expression of cytoprotective heat shock proteins (HSPs), molecular chaperones/cochaperones constituting a major component of the cell protein quality control machinery essential to circumvent stress-induced degradation and aggregation of misfolded proteins. Herein we identify human NF-κB repressing factor (NKRF) as a nucleolar HSP essential for nucleolus homeostasis and cell survival under proteotoxic stress. NKRF acts as a thermosensor translocating from the nucleolus to the nucleoplasm during heat stress; nucleolar pools are replenished during recovery upon HSF1-mediated NKRF resynthesis. Silencing experiments demonstrate that NKRF is an unconventional HSP crucial for correct ribosomal RNA (rRNA) processing and preventing aberrant rRNA precursors and discarded fragment accumulation. These effects are mediated by NKRF interaction with the 5′-to-3′ exoribonuclease XRN2, a key coordinator of multiple pre-rRNA cleavages, driving mature rRNA formation and discarded rRNA decay. Under stress conditions, NKRF directs XRN2 nucleolus/nucleoplasm trafficking, controlling 5′-to-3′ exoribonuclease nucleolar levels and regulating rRNA processing. Our study reveals a different aspect of rRNA biogenesis control in human cells and sheds light on a sophisticated mechanism of nucleolar homeostasis surveillance during stress.