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LABORATORY OF PROTEIN METABOLISM

Wojciech Pokrzywa Lab

Who we are

We are a research lab located at the International Institute of Molecular and Cell Biology in Warsaw, one of the best research institutions in Poland.

We focus on mechanisms of protein metabolism - maintenance of the balance between the synthesis and degradation of proteins. We explore the regulation of translation, the ubiquitin-proteasome system, the chaperone network, and muscular exophers in proteostasis. However, we are sometimes intrigued by topics outside this list.

In Pokrzywa lab, we use a combination of biochemical, microscopic, molecular genetics, and bioinformatics techniques, supported by mammalian cell assays and the nematode C. elegans.

Research

Scheme of impact of cold on transcriptome and proteome
Cellular adaptation to cold

To counteract cold, organisms developed various responses, ranging from cold avoidance to adaptation. The latter strategy is used by hibernating animals, which, in extreme cases, can survive subzero temperatures for many days.
We focus on deciphering mechanisms that alter the abundance and types of cellular messenger RNAs and proteins because these kinds of molecules are critical for live-or-die decisions of the cell. We are also investigating the role of protein quality control networks and the ubiquitin system during C. elegans recovery from cold stress. We also conduct drug screens to identify molecules that support the ability of C. elegans to survive cold stress. 

Scheme of muscular exopheresis
Regulation of exophergenesis

We showed that body wall muscles of C. elegans release exophers that can transport muscle-synthesized yolk proteins to support offspring development, increasing their odds of development and survival (Turek et al., 2021). However, we do not know how exophergenesis is regulated in response to external factors that impact animal development and reproduction.
C. elegans exhibits a range of social behaviors that are primarily governed by various pheromones. Pheromone and sensory neuron-based communication between animals modulates animal growth, generation time, and maternal provisioning, and we explore this system to determine the influence of social cues on exophergenesis. We recently found that exophers are differentially modulated by sex-specific ascarosides (i.e., pheromones that are used in communication between individuals) and that sensory neurons and the G-protein coupled receptor 173 regulate exophergenesis in response to environmental stimuli and pheromones (Banasiak et al., 2023). We also found that AQR/PQR/URX neurons, which are directly exposed to pseudocoelomic fluid and monitor the worm's body interior, restrict muscle exopher production.
To our knowledge, our newest study is the first to report how animal communication influences somatic extracellular vesicle production. We currently explore this model to identify the molecular mechanism of exophergenesis at the muscle level.

Schema of E3 ligase complex
E3 ligase complexes in the regulation of lipid metabolism

The cooperation of E3 ligases (i.e., essential components of the ubiquitin-proteasome system that recognize damaged and unwanted proteins) can lead to the formation of alternative ubiquitination structures that aid in directing substrate specificity. CHIP and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the UPS. CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3 set has remained obscure.
We study the molecular mechanism and function of the CHN-1–UFD-2 complex in C. elegans. Our data show that UFD-2 binding promotes cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, the HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitination and regulate S-adenosylhomocysteinase, a key enzyme in the S-adenosylmethionine regeneration cycle, which is essential for S-adenosylmethionine-dependent methylation. Our results define the molecular mechanism that underlies the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitination. We currently investigate new substrates of the CHN-1-UFD-2 complex that are involved in lipid metabolism.

Scheme of a lysine desert protein
Regulation of lysine-deficient proteome through non-canonical ubiquitination

The ubiquitin-proteasome system (UPS) is a major pathway that removes damaged and unwanted proteins. The proteasome recognizes ubiquitinated proteins – tagged with a small protein named ubiquitin and degrades them. Ubiquitin is mainly attached to lysine residues of the protein destined for degradation in a process termed ubiquitination. However, the UPS must effectively eliminate only undesirable proteins while leaving the functional and essential ones intact. An intuitive mechanism of a proteome susceptible to premature ubiquitination is to avoid lysines in critical domains or entire sequences, potentially leaving a few whose ubiquitination can be precisely controlled.
An extensive lysine-less region (i.e., lysine desert) in the yeast E3 ligase Slx5 was shown to counteract its ubiquitin-dependent turnover. We conducted bioinformatic screens among prokaryotes and eukaryotes to describe the scope and conservation of this phenomenon (Szulc et al., 2023). We found that lysine deserts are widespread among bacteria using pupylation-dependent proteasomal degradation, an analog of the UPS. In eukaryotes, lysine deserts appear with increasing organismal complexity, and the most evolutionarily conserved are enriched in UPS members. Using VHL and SOCS1 E3 ligases, which elongated their lysine desert during the course of evolution, we established that they are non-lysine ubiquitinated, which does not influence their stability and can be subjected to proteasome turnover regardless of ubiquitination. Our data suggest that a combination of non-lysine ubiquitination and ubiquitin-independent degradation may control the function and fate of the lysine-deficient proteome because the presence of lysine deserts does not correlate with half-life. We currently study the regulation of other lysine-depleted receptors of cullin-RING ligases, as well as we aim to decipher the role of lysine-less regions in protein turnover and to develop an analytical method to catalog and study the role of non-lysine ubiquitination by applying biochemical and proteomic approaches and deep learning.

Scheme of myosin assembly
DEGRONOPEDIA: a web server for the proteome-wide inspection of degrons

A degradation-targeting degron comprises a nearby ubiquitin-modified residue and an intrinsically disordered region that interacts with the proteasome. Degron signaling has been studied over recent decades, but there are no resources for the systematic screening of degron sites to facilitate studies on their biological significance, such as targeted protein degradation approaches.
To bridge this gap, we are developing DEGRONOPEDIA (degronopedia.com), a web server that allows the exploration of degron motifs in proteomes of several model organisms and maps these data to lysine, cysteine, threonine, and serine residues that can undergo ubiquitination and to intrinsically disordered regions that are proximal to them, both in sequence and structure. The server provides the evolutionary context of degrons and reports post-translational modifications and pathogenic mutations within the degron and its flanking regions as these can modulate the degron’s accessibility. DEGRONOPEDIA allows analyses of custom sequences/structures to examine them for degron motifs. We also implemented machine learning to predict the stability of protein N- and C -termini, facilitating the identification of substrates of N-/C-degron pathways. This project also concerns the experimental validation of predicted degrons in a cellular context. We are continually implementing new features of DEGRONOPEDIA based on feedback from users and expanding the database of degron motifs because our tool aims to stimulate research on degron signaling. 

News

March 2023 | Ph.D. defense of Aniruddha

Ph.D. defense of Aniruddha

Huge congratulations to Aniruddha on an outstanding Ph.D. dissertation defense!

Aniruddha's research on the synergistic cooperation between the ubiquitin ligases CHN-1 and UFD-2 resulted in a first-author article that landed on the cover of the EMBO Journal and formed the basis of his Ph.D. thesis. During doctoral studies, Aniruddha also presented his results at several international conferences and participated in other projects in the lab. Aniruddha will continue his research on the ubiquitin-proteasome system as a post-doc at the University of Texas at Austin in Prof. Andreas Matouschek's group.

February 2023 | Our presence at the Rare Disease Conference

Aging iron impact

On the occasion of the upcoming Rare Diseases Day, our Ph.D. student Natalia had the pleasure of presenting at the 6th National Scientific Conference on Rare Diseases in the 21st Century our research using the nematode C. elegans, in which we demonstrated that an ultra-rare mutation in the FEM1C gene causes neurodevelopmental disorders.
We hope that our study will contribute to the development of personalized therapies for patients with mutations in FEM1C.

February 2023 | Konrad admitted to the Ph.D. program

Photo of Konrad

Congratulations to our former master's student Konrad on his admission to the Ph.D. program. Konrad will conduct his doctoral research under the supervision of an ERC grantee, Dr. Adam Kłosin, at the Nencki Institute of Experimental Biology. 

Konrad, we are very proud of you and wish you many successes and impactful discoveries!

February 2023 | Celebrating
#WomenInSTEM Day

Natalia's interview panel

Thanks to the EU-LIFE alliance of leading European life sciences research centers, our Ph.D. student Natalia gave an interview about the challenges of ensuring equal opportunities in science on the occasion of the International Day of Women and Girls in Science #WomenInSTEM.
In Pokrzywa lab, we fully support women and promote equality in all aspects.

February 2023 | New article
published

Aging iron impact

Together with the group of Dr. Katarzyna Mleczko-Sanecka from our Institute, we have published an article showing that defects in red pulp macrophages - cells responsible for maintaining blood homeostasis - occur in early aging and disrupt iron turnover, contributing to anemia in elderly age.

We are very proud of this intra-institute collaboration and look forward to more thrilling joint research!

January 2023 | New preprint
published

Lysine desert definitions

We have published a preprint describing our research on lysine-less proteins, so-called lysine deserts. We explored their prevalence, evolutionary conservation, and possible functional roles in prokaryotes and eukaryotes. In addition, using the example of lysine-free variants of VHL and SOCS1 proteins, we showed that they can undergo non-lysine ubiquitination and proteasome-dependent turnover which is independent of their ubiquitination.

January 2023 | New article
published

protocol graphical abstract

We have just published a detailed protocol for quantifying muscle exophers in C. elegans. We hope that our resource will be useful to the scientific community in performing cutting-edge studies of these exciting extracellular vesicles.

December 2022 | New preprint published

exopher graphical abstract

Together with the group of Dr. Michał Turek from the Institute of Biochemistry and Biophysics, we released a preprint where we show how sex-specific pheromones and olfactory neurons regulate muscle extracellular vesicles (exophers) in C. elegans.
Our findings may imply the existence of an analogous mechanism regulating cardiomyocyte exophers, which contributes to the risk of cardiovascular disease in humans.

December 2022 | Our presence at the PhasAGE conference

CHIP protein in nucleolus

We congratulate our senior researcher Małgorzata on giving a talk about nucleolar proteostasis control at the Biomolecular condensates in biology and disease conference organized by the PhasAGE consortium in Brussels. We hope our data shed some light on the relation between CHIP ubiquitin ligase and nucleolus in cellular stress and phase separation.

Publications

2023

Impaired iron recycling from erythrocytes is an early hallmark of aging
Slusarczyk P., Mandal P.K., Zurawska, G. Niklewicz M., Chouhan K., Mahadeva R., Jończy A., Macias M., Szybinska A., Cybulska-Lubak M., Krawczyk O., Herman S., Mikula M., Serwa R., Lenartowicz M., Pokrzywa W. & Mleczko-Sanecka K.
eLife
doi: 10.7554/eLife.79196 

Lysine-deficient proteome can be regulated through non-canonical ubiquitination and ubiquitin-independent proteasomal degradation
Szulc N.A.*, Piechota M., Thapa P., & Pokrzywa W.*
bioRxiv
doi: 10.1101/2023.01.18.524605 

Structural Interaction Fingerprints and Machine Learning for predicting and explaining binding of small molecule ligands to RNA
Szulc N.A.*, Mackiewicz Z., Bujnicki J.M.* & Stefaniak F.*
bioRxiv
doi: 10.1101/2023.01.11.523582 

Preparation of Caenorhabditis elegans for Scoring of Muscle-derived Exophers
Banasiak K.*, Turek M.* & Pokrzywa W.*
Bio-protocol
doi: 10.21769/BioProtoc.4586

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bolded - member of the Pokrzywa lab
* corresponding author
† equal contribution

2022

Pheromone-dependent olfaction bidirectionally regulates muscle extracellular vesicles formation
Banasiak K., Szczepańska A., Kołodziejska K., Tudu Ibrahim A., Pokrzywa W.* & Turek M.*
bioRxiv
doi: 10.1101/2022.12.22.521669 

In silico analysis of the profilaggrin sequence indicates alterations in the stability, degradation route, and intracellular protein fate in filaggrin null mutation carriers
Paul A.A.†, Szulc N.A.†, Kobiela A., Brown S.J., Pokrzywa W.* & Owsiak-Gutowska D.*
Research Square
doi: 10.21203/rs.3.rs-2302890/v1 

A novel de novo FEM1C variant is linked to neurodevelopmental disorder with absent speech, pyramidal signs, and limb ataxia
Dubey A.A.†, Krygier M.†, Szulc N.A.†, Rutkowska K., Kosinska J., Pollak A., Rydzanicz M., Kmiec T., Mazurkiewicz-Beldzinska M., Pokrzywa W.* & Ploski R.*
Human Molecular Genetics
doi: 10.1093/hmg/ddac276 

A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing
Balaji V., Müller L., Lorenz R., Kevei E., Zhang W.H., Santiago U., Gebauer J., Llamas E., Vilchez D., Camacho C.J., Pokrzywa W. & Hoppe T.
Molecular Cell
doi: 10.1016/j.molcel.2022.08.003 

Ferritin-mediated iron detoxification promotes hypothermia survival in Caenorhabditis elegans and murine neurons
Pekec T., Lewandowski J., Komur A.A., Sobańska D., Guo Y., Świtońska-Kurkowska K., Małecki J.M., Dubey A.A., Pokrzywa W, Frankowski M., Figiel M. & Ciosk R.
Nature Communications
doi: 10.1038/s41467-022-32500-z 

A heterotypic assembly mechanism regulates CHIP E3 ligase activity
Das, A., Thapa, P., Santiago, U., Shanmugam, N., Banasiak, K., Dabrowska, K., Nolte, H., Szulc, N.A., Gathungu, R. M., Cysewski, D., Krueger, M., Dadlez, M., Nowotny, M., Camacho, C. J., Hoppe, T., & Pokrzywa, W.*
EMBO Journal
doi: 10.15252/embj.2021109566 

fingeRNAt - a novel tool for high-throughput analysis of nucleic acid-ligand interactions
Szulc, N.A.*, Mackiewicz, Z., Bujnicki, J.M.*, & Stefaniak F.*
PLOS Computational Biology
doi: 10.1371/journal.pcbi.1009783 

DEGRONOPEDIA - a web server for proteome-wide inspection of degrons
Szulc N.A.*, Stefaniak F., Piechota M., Cappannini A., Bujnicki J.M. & Pokrzywa W.*
bioRxiv
doi: 10.1101/2022.05.19.492622 

CHIP ubiquitin ligase is involved in the nucleolar stress management
Piechota M.*, Biriczova L.†, Kowalski K.†, Szulc N.A. & Pokrzywa W.*
bioRxiv
doi: 10.1101/2022.05.17.492288

A novel de novo FEM1C variant is linked to neurodevelopmental disorder with absent speech, pyramidal signs, and limb ataxia
Dubey A.A.†, Krygier M.†, Szulc N.A.†, Rutkowska K., Kosinska J., Pollak A., Rydzanicz M., Kmiec T., Mazurkiewicz-Beldzinska M., Pokrzywa W.* & Ploski R.*
bioRxiv
doi: 10.1101/2022.04.24.489208

Impaired iron recycling from erythrocytes is an early iron-dependent hallmark of aging
Mandal, P. K., Slusarczyk P., Zurawska G., Cybulska M., Krawczyk O., Mikula M., Herman S., Lenartowicz M., Serwa R., Pokrzywa W., & Mleczko-Sanecka K.
bioRxiv
doi: 10.1101/2022.01.16.476518

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bolded - member of the Pokrzywa lab
* corresponding author
† equal contribution

2021

fingeRNAt - a novel tool for high-throughput analysis of nucleic acid-ligand interactions
Szulc, N.A.*, Mackiewicz, Z., Bujnicki, J.M.*, & Stefaniak F.*
bioRxiv
doi: 10.1101/2021.12.23.474073 

Heterotypic assembly mechanism regulates CHIP E3 ligase activity
Das, A., Thapa, P., Santiago, U., Shanmugam, N., Banasiak, K., Dabrowska, K., Nolte, H., Szulc, N.A., Gathungu, R. M., Cysewski, D., Krueger, M., Dadlez, M., Nowotny, M., Camacho, C. J., Hoppe, T., & Pokrzywa, W.*
bioRxiv
doi: 10.1101/2021.08.20.457118 

Muscle-derived exophers promote reproductive fitness
Turek, M., Banasiak, K., Piechota, M., Shanmugam, N., Macias, M., Śliwińska, M. A., Niklewicz, M., Kowalski, K., Nowak, N., Chacinska, A., & Pokrzywa, W.*
EMBO reports
doi: 10.15252/embr.202052071 

Maintaining proteostasis under mechanical stress
Höhfeld, J., Benzing, T., Bloch, W., Fürst, D.O., Gehlert, S., Hesse, M., Hoffmann, B., Hoppe, T., Huesgen, P.F., Köhn, M., Kolanus, W., Merkel, R., Niessen, C. M., Pokrzywa, W., Rinschen, M.M., Wachten, D., & Warscheid, B.
EMBO reports
doi: 10.15252/embr.202152507 

The dose-dependent pleiotropic effects of the UBB+1 ubiquitin mutant
Banasiak, K., Szulc, N.A., & Pokrzywa, W.*
Frontiers in Molecular Biosciences
doi: 10.3389/fmolb.2021.650730

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bolded - member of the Pokrzywa lab
* corresponding author

2020

The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells
Fatima, A., Irmak, D., Noormohammadi, A., Rinschen, M.M., Das, A., Leidecker, O., Schindler, C., Sánchez-Gaya, V., Wagle, P., Pokrzywa, W., Hoppe, T., Rada-Iglesias, A., & Vilchez, D.
Communications Biology
doi: 10.1038/s42003-020-0984-3 

Ubiquitin signaling regulates RNA biogenesis, processing, and metabolism
Thapa, P., Shanmugam, N., & Pokrzywa, W.*
BioEssays
doi: 10.1002/bies.201900171

CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage
Albert, M.-C., Brinkmann, K., Pokrzywa, W., Günther, S. D., Krönke, M., Hoppe, T., & Kashkar, H.
Cell Death & Disease
doi: 10.1038/s41419-020-02923-x 

Bioshell 3.0: Library for processing structural biology data
Macnar, J.M., Szulc, N.A., Kryś, J.D., Badaczewska-Dawid, A.E., & Gront, D.
Biomolecules
doi: 10.3390/biom10030461 

Pathogenic variants in the myosin chaperone UNC-45B cause progressive myopathy with eccentric cores
Donkervoort, S., Kutzner, C. E., Hu, Y., Lornage, X., Rendu, J., Stojkovic, T., Baets, J., Neuhaus, S.B., Tanboon, J., Maroofian, R., Bolduc, V., Mroczek, M., Conijn, S., Kuntz, N. L., Töpf, A., Monges, S., Lubieniecki, F., McCarty, R. M., Chao, K. R., Governali, S., Böhm, J., Boonyapisit, K., Malfatti, E., Sangruchi, T., Horkayne-Szakaly, I., Hedberg-Oldfors, C., Efthymiou, S., Noguchi, S., Djeddi, S., Iida, A., di Rosa, G., Fiorillo, C., Salpietro, V., Darin, N., Faure, J., Houlden, H., Oldfors, A., Nishino, I., de Ridder, W., Straub, V., Pokrzywa, W., Laporte, J., Foley, R., Romero, N.B., Ottenheijm, C., Hoppe, T., & Bönnemann, C.G.
The American Journal of Human Genetics
doi: 10.1016/j.ajhg.2020.11.002

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bolded - member of the Pokrzywa lab
* corresponding author

2018

Ubiquitylation pathways in insulin signaling and organismal homeostasis
Balaji, V.†, Pokrzywa, W.†, & Hoppe, T.
BioEssays
doi: 10.1002/bies.201700223 

The ubiquitin ligase UBR5 suppresses proteostasis collapse in pluripotent stem cells from Huntington’s disease patients
Koyuncu, S., Saez, I., Lee, H. J., Gutierrez-Garcia, R., Pokrzywa, W., Fatima, A., Hoppe, T., & Vilchez, D.
Nature Communications
doi: 10.1038/s41467-018-05320-3

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bolded - member of the Pokrzywa lab
† equal contribution

2017

Chaperone-directed ubiquitylation maintains proteostasis at the expense of longevity
Pokrzywa, W., Lorenz, R., & Hoppe, T.
Worm
doi: 10.1080/21624054.2017.137140 

CHIPped balance of proteostasis and longevity
Pokrzywa, W., & Hoppe, T.
Oncotarget
doi: 10.18632/oncotarget.22101 

Repair or destruction - an intimate liaison between ubiquitin ligases and molecular chaperones in proteostasis
Kevei, É.†, Pokrzywa, W.†, & Hoppe, T.
FEBS Letters
doi: 10.1002/1873-3468.12750

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bolded - member of the Pokrzywa lab
† equal contribution

Facilities

In the Pokrzywa lab, in addition to standard equipment and a coffee machine, we have several state-of-the-art research instruments for worms and proteins analysis, including:

WormLab® Imaging System

ScreenChip™ System

wMicroTracker

CherryTemp

ZEISS Axio Zoom.V16 Fluorescence Microscope

ÄKTA Go Protein Purification System

Funding

Logos of funding agencies
Open grants
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Closed grants
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Team

Principal Investigator

Photo of Dr. Wojciech Pokrzywa

Wojciech Pokrzywa, Ph.D., D.Sc.

ORCID, LinkedIn, Twitter 

During his Ph.D. studies at the Catholic University of Louvain, Belgium, Wojciech Pokrzywa investigated the function of the ubiquitin-proteasome system in regulating membrane protein localization in yeast. In 2009, he joined the laboratory of Prof. Thorsten Hoppe at the University of Cologne, Germany, where he studied the mechanisms of proteostasis during development and aging in C. elegans.
In mid-2017, he started his own research group in Warsaw focusing on the mechanisms of protein metabolism regulation.

Lab Members

Senior Researchers 

Małgorzata Piechota, Ph.D.

POSTDOCTORAL RESEARCHERS

Abhishek Dubey, Ph.D.

Laboratory Support Specialists 

Anna Grabowska, Ph.D.

Marta Niklewicz, M.Sc.

Lilla Biriczová, M.Sc.

Patryk Marcinkowski, M.Sc.

Ph.D. STUDENTS

Pankaj Thapa, M.Sc.

Natalia Szulc, M.Sc.

Anwesha Sarkar, M.Sc.

Join Us

Are you interested in molecular mechanisms of proteostasis? Are you looking for a passionate group of scientists with an exceptionally friendly working atmosphere? Why not join the Pokrzywa lab?

We are currently looking for a postdoctoral researcher.
The deadline for application is 20th March 2023. Interested candidates are encouraged to contact the head of the lab - Wojciech Pokrzywa.

Contact

Principal Investigator

Wojciech Pokrzywa, Ph.D., D.Sc. 

Phone: +48 22 597 07 43
E-mail: wpokrzywa@iimcb.gov.pl

Laboratory Manager

Anna Grabowska, Ph.D.

Phone: +48 22 597 07 77
E-mail: agrabowska@iimcb.gov.pl

Address

Laboratory of Protein Metabolism

International Institute of Molecular and Cell Biology in Warsaw

4 Ks. Trojdena Street

02-109 Warsaw, Poland 

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