Farid El Oualid

UbiQ Bio B.V.

DNA damage tolerance (DDT) is an important pathway that allows our cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA: Monoubiquitination (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as substrate for K63-linked polyubiquitination that leads to HR-mediated bypass mechanisms. Recent wor...

The outcome of cell signaling depends not only on signal strength but also on temporal progression. We use Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to investigate intracellular signaling dynamics. We examined the β1 receptor-Gαs-cAMP signaling axis using both widefield frequency domain FLIM (fdFLIM) and fast...

Emerging evidence suggests that cell signaling outcomes depend not only on the signal strength but also on its temporal progression. Our lab employs Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to study intracellular signaling dynamics. We studied activation of β1 receptors by Isoproterenol, which triggers cAMP...

Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host’s key im...

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct archite...

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed...

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and...

Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein in...

Ubiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the ε-amine of lysine or N-terminal methionine residue of a substrate protein or another Ub molecule. Each of the seven internal lysine residues and the N-terminal methionine residue of Ub can be linked to the...

OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsi...

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5′-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine...

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal...

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal...

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be simila...

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions o...

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activ...

Summary The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe UbG76Dha-Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic sit...

Cruciferous vegetables such as broccoli and kale have well documented chemopreventative and anticancer effects that are attributed to the presence of isothiocyanates (ITCs). ITCs modulate the levels of many oncogenic proteins, but the molecular mechanisms of ITC action are not understood. We previously reported that phenethyl isothiocyanate (PEITC)...

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this...

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regu...

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub...

The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). While many potential targets of ITCs have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report tha...

The chemoprotective effects of a diet rich in broccoli or kale has been appreciated for several decades. Such cruciferous vegetables are a rich source of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). Each of these ITCs have antiproliferative activity against various tumors and PEITC is in clinical trials for lung and o...

Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in R...

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating e...

Protein modification by interferon‐stimulated gene 15 (ISG15), an ubiquitin‐like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin‐specific protease 18 (USP18). To date, analysis of the molecular prop...

We developed a scalable synthesis of [gamma]-thiolysine starting straight from lysine. The application of [gamma]-thiolysine was compared to [small delta]-thiolysine in the chemical synthesis of K48 and K33 linked diubiquitin conjugates. Both [gamma]- and [small delta]-thiolysine were found to perform equally efficiently as handles for non-enzymati...

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types...

Sequence alignments of OTUD1 (A), OTUD2 (B), and OTUD3 (C) catalytic domains as observed in the crystal structure, derived from the Ensembl database (www.ensembl.org). Secondary structure elements for the human sequence are as indicated, the Cys and His loops are shown in pink, catalytic residues are labeled by stars, and S2 site residues in OTUD2...

Values in parentheses are for the highest resolution shell.

A litter of pups: The synthesis and in vitro evaluation of new Pup-based fluorogenic substrates for Dop, the mycobacterial depupylase, are described. A full-length Pup-amidomethylcoumarin conjugate as well as an amino-terminus-truncated analogue exhibited high sensitivity and specificity towards hydrolysis by Dop. The substrates developed here migh...

Thiolysine-mediated chemical ligation has generated fluorescence polarisation assay reagents based on isopeptide-linked ubiquitin-like protein conjugates. These have been used to monitor the activity of ubiquitin(-like) proteases. Thus, it is now possible to generate assay reagents that contain substrate-derived elements around the isopeptide linka...

Post-translational modification of proteins with ubiquitin (Ub) and Ub chains controls numerous biochemical events. Although it has been proven that all Ub-Ub linkages are formed in cells, studies have been limited for a long time to K48 and K63 chains as these can be generated biochemically. Access to the remaining (atypical) Ub-Ub chain types has...

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of mag...

Eight different types of ubiquitin linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are mainly unknown. We here reveal how the human ovarian tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydroly...

Changing the subject: An efficient linear solid-phase peptide synthesis of ubiquitin (Ub) has been developed. This approach allows the incorporation of desired tags and mutations (see picture; blue denotes a pseudoproline dipeptide, red a dimethoxybenzyl dipeptide) as well as specific C-terminal modification and the construction of all diubiquitin...

We demonstrate that oxime ligation is an efficient, straightforward, and generally applicable strategy for generating nonhydrolyzable ubiquitin (Ub)-isopeptide isosteres. We synthesized nonhydrolyzable K48- and K63-linked Ub-isopeptide isosteres to investigate the selectivity of deubiquitinating enzymes for specific linkages employing surface plasm...

Die Einführung einer Thiolgruppe in Kombination mit einer chemoselektiven Ligation unter Bildung eines Disulfid-verbrückten Biokonjugats ist eine selektive und nützliche Methode zur ortsselektiven Proteinglycosylierung. Die Phosphan-vermittelte Entschwefelung solcher Glycokonjugate zu reduktionsbeständigen Thioether-verbrückten Analoga vervollständ...

(Chemical Presented) Strengthening the bond: The introduction of a thiol tag in combination with chemoselective ligation to form a disulfide-linked bioconjugate is a selective and useful method for site-selective protein glycosylation. The phosphine-mediated desulfurization of such glycoconjugates to their reductant-resistant thioether-linked count...

Single-walled carbon nanotubes (SWNTs) functionalized with organic molecules bearing high-scattering element tags have allowed direct visualization of modification on the atomic scale. The inclusion of these tags in protecting groups allowed their subsequent removal and reincorporation to allow visualization of corresponding protection and deprotec...

The enzyme protein:geranylgeranyl transferase-1 (PGGT-1 or GGTase-I) catalyzes the geranylgeranylation of cysteine residues near the C-termini of a variety of proteins, including most monomeric GTP binding precursor proteins belonging to the Rho, Rac and Rap subfamilies. These proteins are involved in signaling pathways controlling important proces...

A combinatorial synthesis of oligopeptide analogues and their evaluation as protein:geranylgeranyl transferase inhibitors is presented. The combinatorial strategy is based on the random mutation, in each new generation, of one of any of the four amino acid building blocks of which the most effective compounds of the previous generation are assemble...

Proefschrift Universiteit Leiden. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

The prenyl transferases protein:farnesyl transferase (PFT) and protein:geranylgeranyl transferase-1 (PGGT-1) are responsible for the post-translational modification of small Gproteins. This post-translational modification comprises the attachment of an isoprenoid (farnesyl or gerangeranyl) to the C-terminus of the G-protein and is essential for a p...

Ca(1)a(2)L analogues, having the central dipeptide a(1)a(2) replaced by a sugar amino acid, were provided at the N-terminal end directly or via a spacer with a lipid. The inhibitory potency toward PGGT-1 of the set of lipophilic Ca(1)a(2)L analogues was improved in comparison with the original analogues, 1 and 2. The most potent inhibitors, 39 and...

A practical gram-scale and high-yielding synthesis of the antimicrobial peptide gramicidin S is presented. An Fmoc-based solid-phase peptide synthesis protocol is employed for the generation of the linear decapeptide precursor, which is cyclized in solution to afford the target compound. The versatility of our method is demonstrated by the construc...

[reaction: see text] Novel highly functionalized dipeptide isosters are synthesized via a diastereoselective alkyl/arylation protocol of a glucose-derived (R)-tert-butanesulfinylimine. One of these novel sugar amino acid derivatives, a D-Ala-Ser/Thr isostere, was applied in a peptide synthesis protocol to afford a cyclic tetramer.