Biology subjects

Kleifeld, O.

Publications and source records attributed to Kleifeld, O..

12 recordsLinked to original sources

bioRxiv2025

The GT1 domain of RNase J ensures RNA quality control through dsRNA binding in Arabidopsis plastids

RNase J is a ribonuclease found in bacteria, archaea, and plant chloroplasts, and plays diverse roles in RNA maturation and stability. Chloroplast RNase J is encoded by the nuclear RNJ locus and is essential for embryo maturation. Arabidopsis or tobacco plants depleted for RNase J accumulate massive amounts of double-stranded RNA, which interferes with translation and causes chlorosis. Land plant RNase J uniquely contains a C-terminal GT1 domain, a DNA- binding motif found in transcription factors. Here, we have used complementation of an Arabidopsis rnj mutant with versions of RNase J with a mutated or deleted GT1 domain to investigate its role in RNase J function. We show that in vitro, the recombinant GT1 domain binds both double-stranded RNA and DNA, but not single-stranded nucleic acids, with no sequence specificity. Furthermore, while RNase J lacking GT1 binding complements the rnj mutant, these plants accumulate high levels of dsRNA as detected by immunolocalization and RNA-Seq. GT1 mutations also change RNase J solubility in vivo, suggesting that the GT1 domain is involved in localization within the plastid. Taken together, our results suggest that the GT1 domain plays a key role in dsRNA removal through localizing the enzyme and/or selectively binding the dsRNA substrate. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/682605v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@e5642borg.highwire.dtl.DTLVardef@136456org.highwire.dtl.DTLVardef@1205466org.highwire.dtl.DTLVardef@184f72b_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

USP15: the fourth Proteasome-associated DUB

The human genome encodes approximately 100 deubiquitinating enzymes (DUBs), but only three are considered proteasome-associated DUBs (pDUBs): PSMD14/Rpn11, USP14, and UCHL5. Among these, only PSMD14 is an integral 19S subunit, whereas USP14 and UCHL5 bind transiently to specific proteasomal subunits. Given the dynamic nature of proteasome composition, we searched for additional pDUBs. USP15 was found to be associated with 26S proteasomes purified from cultured cells. In proteasome preparations from erythrocytes, USP15 was identified as the most abundant transient pDUB. It was even feasible to separate proteasomes containing USP15 from those containing USP14. Although USP15 utilizes an internal ubiquitin-like (UBL) domain for positioning itself at the proteasome, it did not compete with the UBL-containing USP14. USP15 facilitated substrate selection at the proteasome by efficiently disassembling short polyubiquitin (polyUb) chains, while sparing K48-linked tetra-ubiquitin conjugates from deubiquitination. This feature may aid the proteasome in differentiating between substrates to be rescued from those committed to proteolysis. Identification of a fourth pDUB encourages the continued search for additional proteasome-interacting proteins that modulate its substrate specificity in a context-specific manner.

biochemistry↗

bioRxiv2024

Oceanic photosynthesis is directly affected by cyanophage NblA proteins

Marine picocyanobacteria are abundant photosynthetic organisms of global importance. They coexist in the ocean with cyanophages, viruses that infect cyanobacteria. Cyanophages carry many auxiliary metabolic genes acquired from their hosts that are thought to redirect host metabolism for the phages benefit1-5. One such gene is nblA which is present in multiple cyanophage families2,6-8. Under nutrient deprivation the cyanobacterial NblA is responsible for inducing proteolytic degradation of the phycobilisome9-11, the large cyanobacterial photosynthetic light harvesting complex. This increases the pool of amino acids available for essential tasks11, serving as a survival mechanism12. Ectopic expression of different cyanophage nblA genes results in host pigment protein degradation9,6. However, the benefit of the cyanophage-encoded NblA for the cyanophage and the broader impact on the host are unknown. Here, using a recently developed genetic manipulation system for marine cyanophages14, we reveal that cyanophage NblA significantly accelerates the cyanophage infection cycle, directs degradation of the host phycobilisome and other proteins and reduces host photosynthetic light harvesting efficiency. Metagenomic analysis revealed that cyanophages carrying nblA are widespread in the oceans and compose 35% and 65% of oceanic T7-like cyanophages in the surface and deep photic zones, respectively. Our results show a large benefit of the nblA gene to the cyanophage while exerting a negative effect on the host photosynthetic apparatus and host photosynthesis. These findings suggest that cyanophage nblA has an adverse global impact on light harvesting by oceanic picocyanobacteria.

microbiology↗

bioRxiv2024

The high-light-tolerant desert alga Chlorella ohadii is protected from oxidative stress by a non-photochemical quenching-independent mechanism

Non-photochemical quenching (NPQ) mechanisms are crucial for protecting photosynthesis from photoinhibition in plants, algae, and cyanobacteria, and their modulation is a long-standing goal for improving photosynthesis and crop yields. The current work demonstrates that Chlorella ohadii, a green micro-alga that thrives in the desert under high light intensities which are fatal to many photosynthetic organisms, does not perform nor require NPQ to protect photosynthesis under constant high light. Instead of dissipating excess energy, it minimizes its uptake by eliminating the photosynthetic antenna of photosystem II. In addition it accumulates antioxidants that neutralize harmful reactive oxygen species (ROS) and ramps up cyclic electron flow around PSI. These NPQ-independent responses proved efficient in preventing ROS accumulation and reducing oxidative damage to proteins in high-light-grown cells.

plant biology↗

bioRxiv2023

Computational design of Matrix Metalloprotenaise-9 (MMP-9) resistant to auto-cleavage

Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix and has been implicated as a major driver in cancer metastasis. Hence, there is a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9Cat) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9Cat variant that is active but stable to autocleavage. For this purpose, we first identified potential autocleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the autocleavage site by predicting mutations that minimize autocleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after seven days of incubation at 37{degrees}C. This MMP-9Cat variant, with an identical to MMP- 9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9CAT stabilization could be applied to redesign of other proteases to improve their stability for various biotechnological applications.

biochemistry↗

bioRxiv2023

Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (And Inactive) Proteasome species

Proteasomes are multi-subunit, multi-catalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast, and the changes these complexes undergo upon the deletion of Pre9 ({Delta}3) or of Sem1 ({Delta}Sem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of non-mature and therefore inactive proteasome protease subunits {beta}2 and {beta}5 in the fully assembled proteasomes. Significance StatementProteasomes, essential protein complexes in eukaryotic cells, degrade misfolded, damaged, or unstructured proteins. Here we present an activity-guided proteomic method to characterize the composition and abundance of proteasomes. When applied to yeast proteasomes, this method revealed discrepancies between proteasome distributions determined by mass spectrometry and peptidase activity. This implies that a substantial portion of the proteasomes may exhibit reduced activity. Our findings indicate that these changes in proteasome activity could be linked to proteasome inhibition by Fub1. Furthermore, we identified signature peptides that indicate incomplete maturation of some of the {beta}2 and {beta}5 proteolytic subunits in fully assembled proteasomes, suggesting that proteasome core particle assembly can proceed even without the complete maturation of all {beta} subunits.

biochemistry↗

bioRxiv2022

In-depth characterization of apoptosis N-terminome reveals a link between caspase-3 cleavage and post-translational N-terminal acetylation

The N-termini of proteins contain information about their biochemical properties and functions. These N-termini can be processed by proteases and can undergo other co- or post-translational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of -amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-terminomic method to study caspase-3 mediated proteolysis both in vitro and during apoptosis in cells. This has enabled us to identify many unreported caspase-3 cleavages, some of which cannot be identified by other methods. Moreover, we have found direct evidence that neo-N-termini generated by caspase-3 cleavage can be further modified by Nt-acetylation. Some of these neo-Nt-acetylation events occur in the early phase of the apoptotic process and may have a role in translation inhibition. This has provided a comprehensive overview of the caspase-3 degradome and has uncovered previously unrecognized crosstalk between post-translational Nt-acetylation and caspase proteolytic pathways.

biochemistry↗

bioRxiv2022

Helicobacter pylori vacuolating cytotoxin A exploits human endosomes for intracellular activation

Helicobacter pylori infection is the main cause of gastric cancer. Vacuolating cytotoxin A (VacA) is a H. pylori pore-forming toxin and a key determinant of gastric cancer risk. VacA is secreted as an 88-kDa polypeptide (p88) that upon interaction with host cells induces cytotoxic effects, including cell vacuolation and mitochondrial dysfunction. These effects are currently believed to be due to VacA p88 accumulating inside host cells and forming oligomeric anion-specific channels in membranes of intracellular compartments. However, the molecular nature of intracellular VacA channels in host cells remains undefined. Here we show that VacA p88 does not accumulate inside human epithelial cells, but instead is rapidly processed in endosomes into smaller p31/p28 and p37 products in a manner that precedes VacA-induced vacuolation. VacA processing requires endosomal acidification and concerted cleavage by multiple endo-lysosomal proteases including cathepsins. In situ structural mapping reveals that upon processing, the toxins central hydrophilic linker and globular C-terminus are excised, whereas oligomerization determinants are retained. Congruently, the processed products are constituents of a high-molecular-weight complex inside the host cell [boxh] which we propose is the intracellular, mature and active VacA pore. These findings suggest that VacA exploits human endosomes for proteolytic processing and intracellular activation. Significance StatementHelicobacter pylori is a cancer-causing bacterium that infects the stomach of billions of people worldwide. Vacuolating cytotoxin A (VacA) is an important H. pylori virulence factor and its activity directly correlates with gastric carcinogenesis. Yet despite decades of intense research, the mechanisms underlying VacA activity in human cells remain incompletely understood. Here, we present evidence suggesting that VacA is activated inside human cells by multi-step proteolytic processing involving endo-lysosomal proteases including cathepsins. We also track and identify the functional processed VacA isoforms in host cells. These results revolutionize our understanding of the mechanism of VacA activation in human cells, whilst expanding our knowledge of the diversity of microbial virulence factors that exploit human endo-lysosomes for pathogenesis.

molecular biology↗

bioRxiv2022

Infection cycle and phylogeny of the Polinton-like virus Phaeocystis globosa virus virophage-14T

Virophages are small dsDNA viruses dependent on a nucleocytoplasmic large-DNA virus infection of a cellular host for replication. Putative virophages infecting algal hosts are classified together with polinton-like viruses, transposable elements widely found in algal genomes, yet the lack of isolated strains raises questions about their existence as independent entities. In this work we isolated and characterized a virophage (PgVV-14T) co-infecting Phaeocystis globosa with the Phaeocystis globosa virus-14T (PgV-14T). PgVV-14T decreases the fitness of its PgV-14T viral host, yet it does not salvage the cellular host population. We found viral-like elements resembling PgVV-14T in Phaeocystis genomes, suggesting that these virophages are capable of integrating to the cellular host genome, bridging the gap between Polinton-like viruses and virophages. This system, with a giant virus, a virophage and endogenous viral elements preying on an algal host, presents an opportunity to gain a better understanding on the evolution of eukaryotes and their viruses.

microbiology↗

bioRxiv2022

N-Terminomic Changes of Neurons During Excitotoxicity Reveals Proteolytic Events Associated with Synaptic Dysfunctions and Potential Targets for Neuroprotection

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase II{beta} (CaMKII{beta}). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKII, an isoform of CaMKII{beta} undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection. In BriefAmeen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection. HighlightsO_LIIdentification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity. C_LIO_LIThe calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons. C_LIO_LIThese pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death. C_LIO_LISome of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection. C_LI Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses(A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKII and CaMKII{beta}, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotection O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/484119v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1c69873org.highwire.dtl.DTLVardef@142db3forg.highwire.dtl.DTLVardef@481521org.highwire.dtl.DTLVardef@6359e2_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryProteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.

neuroscience↗

bioRxiv2020

An Atlas of Phosphorylation and Proteolytic Processing Events During Excitotoxic Neuronal Death Reveals New Therapeutic Opportunities

Excitotoxicity, a neuronal death process in neurological disorders, is initiated by over-stimulation of neuronal ionotropic glutamate receptors. The over-stimulated receptors dysregulate proteases, protein kinases and phosphatases, which in turn modify target neuronal proteins to induce cell death. To decipher this cell death mechanism, we used quantitative proteomics, phosphoproteomics and N-terminomics to identify modified proteins in excitotoxic neurons. Data, available in ProteomeXchange (identifiers: PXD019527 and PXD019211), enabled us to identify over one thousand such proteins with calpains, cathepsins and over twenty protein kinases as their major modifiers. These protein modification events can potentially perturb signalling pathways governing cell survival, synaptogenesis, axonal guidance and mRNA processing. Importantly, blocking the modification of Src protein kinase, a signalling hub in excitotoxic neurons, protected against neuronal loss in vivo in a rat model of neurotoxicity. Besides offering new insights into excitotoxic neuronal death mechanism, our findings suggest potential neuroprotective therapeutic targets for treating neurological disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/151456v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f0191forg.highwire.dtl.DTLVardef@17ba2org.highwire.dtl.DTLVardef@15c0848org.highwire.dtl.DTLVardef@123d28b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMulti-dimensional proteomic analysis identified proteins modified by proteolysis and altered phosphorylation in neurons undergoing excitotoxic cell death. C_LIO_LICalpains, cathepsins and over twenty protein kinases are major modifiers of these proteins. C_LIO_LIThese protein modification events are predicted to impact cell survival, axonal guidance, synaptogenesis and mRNA processing. C_LIO_LIBlocking modification of an identified protein Src, which acts as a major signalling hub in neurons, was protective against excitotoxic injury in vivo. C_LI In BriefUsing multidimensional proteomic approaches, Ameen, et al. mapped the changes of proteome, phosphoproteome and N-terminome of cultured primary neurons during excitotoxicity, a crucial neuronal death process in neurological disorders. These proteomic changes document new excitotoxicity-associated molecular events, and offer insights into how these events are organized to induce neuronal death. Potential therapeutic relevance of these molecular events is illustrated by the demonstration that in vivo blockade of one of these events could protect against excitotoxic neuronal loss.

biochemistry↗

bioRxiv2019

Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex- the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

biochemistry↗