Wiktor Prus, Ph.D., Eng.

Department of Biosystems and Soft Matter (ZBiMM)
Division of Modelling in Biology and Medicine (PMBM)
position: assistant professor
telephone: (+48) 22 826 12 81 ext.: 448
room: 321
e-mail: wprus
personal site: http://pmbm.ippt.pan.pl/web/Wiktor_Prus

Recent publications
1.Czerkies M., Korwek Z., Prus W., Kochańczyk M., Jaruszewicz-Błońska J., Tudelska K., Błoński S., Kimmel M., Brasier A.R., Lipniacki T., Cell fate in antiviral response arises in the crosstalk of IRF, NF-κB and JAK/STAT pathways, Nature Communications, ISSN: 2041-1723, DOI: 10.1038/s41467-017-02640-8, Vol.9, pp.493, 2018
Abstract:

The innate immune system processes pathogen-induced signals into cell fate decisions. How information is turned to decision remains unknown. By combining stochastic mathematical modelling and experimentation, we demonstrate that feedback interactions between the IRF3, NF-κB and STAT pathways lead to switch-like responses to a viral analogue, poly(I:C), in contrast to pulse-like responses to bacterial LPS. Poly(I:C) activates both IRF3 and NF-κB, a requirement for induction of IFNβ expression. Autocrine IFNβ initiates a JAK/STAT-mediated positive-feedback stabilising nuclear IRF3 and NF-κB in first responder cells. Paracrine IFNβ, in turn, sensitises second responder cells through a JAK/STAT-mediated positive feedforward pathway that upregulates the positive-feedback components: RIG-I, PKR and OAS1A. In these sensitised cells, the ‘live-or-die’ decision phase following poly(I:C) exposure is shorter—they rapidly produce antiviral responses and commit to apoptosis. The interlinked positive feedback and feedforward signalling is key for coordinating cell fate decisions in cellular populations restricting pathogen spread.

Keywords:

cellular signalling networks, innate immunity, regulatory networks, stochastic modelling

Affiliations:
Czerkies M.-IPPT PAN
Korwek Z.-IPPT PAN
Prus W.-IPPT PAN
Kochańczyk M.-IPPT PAN
Jaruszewicz-Błońska J.-IPPT PAN
Tudelska K.-IPPT PAN
Błoński S.-IPPT PAN
Kimmel M.-Rice University (US)
Brasier A.R.-University of Texas Medical Branch (US)
Lipniacki T.-IPPT PAN
2.Tudelska K., Markiewicz J., Kochańczyk M., Czerkies M., Prus W., Korwek Z., Abdi A., Błoński S., Kaźmierczak B., Lipniacki T., Information processing in the NF-κB pathway, Scientific Reports, ISSN: 2045-2322, DOI: 10.1038/s41598-017-16166-y, Vol.7, pp.15926, 2017
Abstract:

The NF-κB pathway is known to transmit merely 1 bit of information about stimulus level. We combined experimentation with mathematical modeling to elucidate how information about TNF concentration is turned into a binary decision. Using Kolmogorov-Smirnov distance, we quantified the cell’s ability to discern 8 TNF concentrations at each step of the NF-κB pathway, to find that input discernibility decreases as signal propagates along the pathway. Discernibility of low TNF concentrations is restricted by noise at the TNF receptor level, whereas discernibility of high TNF concentrations it is restricted by saturation/depletion of downstream signaling components. Consequently, signal discernibility is highest between 0.03 and 1 ng/ml TNF. Simultaneous exposure to TNF or LPS and a translation inhibitor, cycloheximide, leads to prolonged NF-κB activation and a marked increase of transcript levels of NF-κB inhibitors, IκBα and A20. The impact of cycloheximide becomes apparent after the first peak of nuclear NF-κB translocation, meaning that the NF-κB network not only relays 1 bit of information to coordinate the all-or-nothing expression of early genes, but also over a longer time course integrates information about other stimuli. The NF-κB system should be thus perceived as a feedback-controlled decision-making module rather than a simple information transmission channel.

Keywords:

cellular signaling networks, innate immunity, stress signaling

Affiliations:
Tudelska K.-IPPT PAN
Markiewicz J.-IPPT PAN
Kochańczyk M.-IPPT PAN
Czerkies M.-IPPT PAN
Prus W.-IPPT PAN
Korwek Z.-IPPT PAN
Abdi A.-New Jersey Institute of Technology (US)
Błoński S.-IPPT PAN
Kaźmierczak B.-IPPT PAN
Lipniacki T.-IPPT PAN
3.Góral A., Bieganowski P., Prus W., Krzemień-Ojak Ł., Kądziołka B., Fabczak H., Filipek A., Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone, PLOS ONE, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0156507, Vol.11, No.6, pp.e0156507-1-18, 2016
Abstract:

The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.

Keywords:

Chaperone proteins, Luciferase, Phosphatases, Enzyme-linked immunoassays, Recombinant proteins, Ligation assay, Plasmid construction, Luciferase assay

Affiliations:
Góral A.-other affiliation
Bieganowski P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Prus W.-other affiliation
Krzemień-Ojak Ł.-other affiliation
Kądziołka B.-other affiliation
Fabczak H.-Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Filipek A.-Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
4.Korwek Z., Tudelska K., Nałęcz-Jawecki P., Czerkies M., Prus W., Markiewicz J., Kochańczyk M., Lipniacki T., Importins promote high-frequency NF-κB oscillations increasing information channel capacity, Biology Direct, ISSN: 1745-6150, DOI: 10.1186/s13062-016-0164-z, Vol.11, No.61, pp.1-21, 2016
Abstract:

BACKGROUND:
Importins and exportins influence gene expression by enabling nucleocytoplasmic shuttling of transcription factors. A key transcription factor of innate immunity, NF-κB, is sequestered in the cytoplasm by its inhibitor, IκBα, which masks nuclear localization sequence of NF-κB. In response to TNFα or LPS, IκBα is degraded, which allows importins to bind NF-κB and shepherd it across nuclear pores. NF-κB nuclear activity is terminated when newly synthesized IκBα enters the nucleus, binds NF-κB and exportin which directs the complex to the cytoplasm. Although importins/exportins are known to regulate spatiotemporal kinetics of NF-κB and other transcription factors governing innate immunity, the mechanistic details of these interactions have not been elucidated and mathematically modelled.
RESULTS:
Based on our quantitative experimental data, we pursue NF-κB system modelling by explicitly including NF-κB-importin and IκBα-exportin binding to show that the competition between importins and IκBα enables NF-κB nuclear translocation despite high levels of IκBα. These interactions reduce the effective relaxation time and allow the NF-κB regulatory pathway to respond to recurrent TNFα pulses of 45-min period, which is about twice shorter than the characteristic period of NF-κB oscillations. By stochastic simulations of model dynamics we demonstrate that randomly appearing, short TNFα pulses can be converted to essentially digital pulses of NF-κB activity, provided that intervals between input pulses are not shorter than 1 h.
CONCLUSIONS:
By including interactions involving importin-α and exportin we bring the modelling of spatiotemporal kinetics of transcription factors to a more mechanistic level. Basing on the analysis of the pursued model we estimated the information transmission rate of the NF-κB pathway as 1 bit per hour.

Keywords:

Karyopherins, Nucleocytoplasmic transport, Negative feedback, Channel information capacity, Mathematical modelling

Affiliations:
Korwek Z.-IPPT PAN
Tudelska K.-other affiliation
Nałęcz-Jawecki P.-University of Warsaw (PL)
Czerkies M.-IPPT PAN
Prus W.-IPPT PAN
Markiewicz J.-IPPT PAN
Kochańczyk M.-IPPT PAN
Lipniacki T.-IPPT PAN
5.Prus W., Żabka M., Bieganowski P., Filipek A., Nuclear translocation of Sgt1 depends on its phosphorylation state, INTERNATIONAL JOURNAL OF BIOCHEMISTRY AND CELL BIOLOGY, ISSN: 1357-2725, DOI: 10.1016/j.biocel.2011.08.010, Vol.43, pp.1747-1753, 2011
Abstract:

Recently we have shown that the Sgt1 (suppressor of G2 allele of Skp1) protein translocates to the nucleus due to heat shock and that the Ca2+-bound form of S100A6 is required for Sgt1 translocation (Prus and Filipek, 2010). In this work we studied the influence of Sgt1 phosphorylation on nuclear translocation. By means of two-dimensional (2D) electrophoresis we showed that in the protein extract of heat-shocked human epidermoid carcinoma (HEp-2) cells a higher level of a basic, most probably non-phosphorylated, form of Sgt1 can be detected. Also, we found a more efficient translocation of Sgt1 induced by heat shock when casein kinase II inhibitor was added to the cells. To confirm the role of Sgt1 phosphorylation/dephosphorylation in its nuclear translocation we transfected cells with non-phosphorylable Sgt1 mutants (S249A, S299A, S249/299A) or a phosphorylation mimic S299D mutant. We found that the levels of S299A and S249/299A mutants were higher than the level of wild type Sgt1 in the nuclear fraction after heat shock. Accordingly, we found that the 139–333 fragment of Sgt1 harboring the mutated residues, but not the 1–138 fragment, translocated to the nucleus upon heat shock. Moreover, we show that S100A6 is required for translocation of the non-phosphorylable Sgt1 mutants and that upon heat shock S100A6 translocates to the nucleus together with Sgt1. In addition, we found that non-phosphorylable Sgt1 mutant interacts with S100A6 more efficiently and at the same time exhibits lower affinity for Hsp90 (heat shock protein 90) than wild type Sgt1. Altogether, our results suggest that S100A6-Ca2+-mediated Sgt1 dephosphorylation promotes its nuclear translocation, most likely due to disruption of the Sgt1-Hsp90 complex.

Keywords:

Sgt1, Protein phosphorylation, Nuclear translocation, S100A6, Heat shock proteins

Affiliations:
Prus W.-other affiliation
Żabka M.-other affiliation
Bieganowski P.-Mossakowski Medical Research Centre, Polish Academy of Sciences (PL)
Filipek A.-Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
6.Prus W., Filipek A., S100A6 mediates nuclear translocation of Sgt1: a heat shock-regulated protein, AMINO ACIDS, ISSN: 0939-4451, DOI: 10.1007/s00726-010-0526-2, Vol.41, pp.781-787, 2011
Abstract:

Sgt1 was originally identified in yeast as a suppressor of the Skp1 protein. Later, it was found that Sgt1 is present in plant and mammalian organisms and that it binds other ligands such as S100A6, a calcium-binding protein. In this work we show that in HEp-2 cells Sgt1 translocates to the nucleus due to heat shock. We also found that in HEp-2 cells with diminished level of S100A6, due to stable transfection with siRNA against S100A6, such translocation occurred at a much smaller scale in comparison with cells expressing a normal level of S100A6. Moreover, translocation of Sgt1 was observed in HEp-2 cells treated with thapsigargin instead of heat shock. In contrast thapsigargin was ineffective in cells with diminished level of S100A6. Thus, our results suggest that increase in intracellular concentration of Ca2+, transduced by S100A6, is necessary for nuclear translocation of the Sgt1 protein.

Keywords:

Sgt1, S100A6 (calcyclin), Heat shock proteins, Nuclear translocation

Affiliations:
Prus W.-other affiliation
Filipek A.-Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
7.Żabka M., Leśniak W., Prus W., Kuźnicki J., Filipek A., Sgt1 has co-chaperone properties and is up-regulated by heat shock, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ISSN: 0006-291X, DOI: 10.1016/j.bbrc.2008.03.055, Vol.370, pp.179-183, 2008
Abstract:

The Sgt1 protein is a binding partner of heat shock proteins such as Hsp90, Hsp70 or Hsc70. In this work we show that the level of Sgt1 is increased in HEp-2 cells exposed to heat shock or radicicol. The citrate synthase aggregation assay shows that Sgt1 attenuates aggregation of the enzyme induced by increased temperature as efficiently as p23, a known co-chaperone of Hsp90. We have cloned two fragments of the human Sgt1 gene promoter (−708/+98 and −351/+98) into pGL3-luciferase vector and found that both fragments generated a 2-fold increase in luciferase activity upon heat shock. Furthermore, electrophoretic mobility shift assay revealed binding of the HSF-1 transcription factor to the heat shock element in the proximal (−42/−2) Sgt1 gene promoter fragment. These results indicate that Sgt1 is a co-chaperone protein with an expression pattern matching that of the well known heat shock proteins.

Keywords:

Sgt1, Heat shock, Radicicol, Sgt1 gene promoter, HSF-1, Co-chaperone

Affiliations:
Żabka M.-other affiliation
Leśniak W.-other affiliation
Prus W.-other affiliation
Kuźnicki J.-other affiliation
Filipek A.-Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
8.Szawłowska U., Prus W., Bielawski W., The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.), ACTA PHYSIOLOGIAE PLANTARUM, ISSN: 0137-5881, Vol.28, pp.517-524, 2006
Abstract:

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seed lings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophobic chromatography on Phenyl- Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units•mg-1of protein with recovery yield of 3 %. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is com posed of four subunits. The enzyme was specific for proline -naphtylamide among various amino acid -naphtylamides.
An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.

Keywords:

proline iminopeptidase, exopeptidases, purification, characteristics, rye

Affiliations:
Szawłowska U.-other affiliation
Prus W.-other affiliation
Bielawski W.-other affiliation