Exposing the small protein load of bacterial life.

The ever-growing repertoire of genomic techniques continues to expand our understanding of the true diversity and richness of prokaryotic genomes. Riboproteogenomics laid the foundation for dynamic studies of previously overlooked genomic elements. Most strikingly, bacterial genomes were revealed to harbor robust repertoires of small open reading frames (sORFs) encoding a diverse and broadly expressed range of small proteins, or sORF-encoded polypeptides (SEPs). In recent years, continuous efforts led to great improvements in the annotation and characterization of such proteins, yet many challenges remain to fully comprehend the pervasive nature of small proteins and their impact on bacterial biology. In this work, we review the recent developments in the dynamic field of bacterial genome reannotation, catalog the important biological roles carried out by small proteins and identify challenges obstructing the way to full understanding of these elusive proteins.

Virotrap: Trapping Protein Complexes in Virus-Like Particles.

The discovery of protein-protein interactions can provide crucial information on protein function by linking proteins into known pathways or complexes within the cell. Mass spectrometry (MS)-based methods, such as affinity purification (AP)-MS and proximity-dependent biotin identification (BioID), allowed for a vast increase in the number of reported protein complexes. As a more recent addition to the arsenal of MS-based methods, Virotrap represents a unique technology that benefits from the specific properties of the human immunodeficiency virus-1 (HIV-1) Gag polyprotein. More specifically, Virotrap captures protein complexes in virus-like particles budded from human embryonic kidney (HEK293T) cells, bypassing the need for cell lysis and thus supporting identification of their content using MS. Being intrinsically different to its two main predecessors, affinity purification MS (AP-MS) and biotin-dependent identification (BioID), Virotrap was shown to complement data obtained with the existing MS-based toolkit. The proven complementarity of these MS-based strategies underlines the importance of using different techniques to enable comprehensive mapping of protein-protein interactions (PPIs). In this chapter, we provide a detailed overview of the Virotrap protocol to screen for PPIs using a bait protein of interest.

Shift in vacuolar to cytosolic regime of infecting Salmonella from a dual proteome perspective.

By applying dual proteome profiling to Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters with its epithelial host (here, S. Typhimurium infected human HeLa cells), a detailed interdependent and holistic proteomic perspective on host-pathogen interactions over the time course of infection was obtained. Data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows, especially in identifying the downregulated bacterial proteome response during infection progression by permitting quantification of low abundant bacterial proteins at early times of infection when bacterial infection load is low. S. Typhimurium invasion and replication specific proteomic signatures in epithelial cells revealed interdependent host/pathogen specific responses besides pointing to putative novel infection markers and signalling responses, including regulated host proteins associated with Salmonella-modified membranes.

Gasdermin D independent canonical inflammasome responses cooperate with caspase-8 to establish host defense against gastrointestinal Citrobacter rodentium infection.

Citrobacter rodentium is an enteropathogen that causes intestinal inflammatory responses in mice reminiscent of the pathology provoked by enteropathogenic and enterohemorrhagic Escherichia coli infections in humans. C. rodentium expresses various virulence factors that target specific signaling proteins involved in executing apoptotic, necroptotic and pyroptotic cell death, suggesting that each of these distinct cell death modes performs essential host defense functions that the pathogen aims to disturb. However, the relative contributions of apoptosis, necroptosis and pyroptosis in protecting the host against C. rodentium have not been elucidated. Here we used mice with single or combined deficiencies in essential signaling proteins controlling apoptotic, necroptotic or pyroptotic cell death to reveal the roles of these cell death modes in host defense against C. rodentium. Gastrointestinal C. rodentium infections in mice lacking GSDMD and/or MLKL showed that both pyroptosis and necroptosis were dispensable for pathogen clearance. In contrast, while RIPK3-deficient mice showed normal C. rodentium clearance, mice with combined caspase-8 and RIPK3 deficiencies failed to clear intestinal pathogen loads. Although this demonstrated a crucial role for caspase-8 signaling in establishing intestinal host defense, Casp8-/-Ripk3-/- mice remained capable of preventing systemic pathogen persistence. This systemic host defense relied on inflammasome signaling, as Casp8-/-Ripk3-/- mice with combined caspase-1 and -11 deletion succumbed to C. rodentium infection. Interestingly, although it is known that C. rodentium can activate the non-canonical caspase-11 inflammasome, selectively disabling canonical inflammasome signaling by single caspase-1 deletion sufficed to render Casp8-/-Ripk3-/- mice vulnerable to C. rodentium-induced lethality. Moreover, Casp8-/-Ripk3-/- mice lacking GSDMD survived a C. rodentium infection, suggesting that pyroptosis was not crucial for the protective functions of canonical inflammasomes in these mice. Taken together, our mouse genetic experiments revealed an essential cooperation between caspase-8 signaling and GSDMD-independent canonical inflammasome signaling to establish intestinal and systemic host defense against gastrointestinal C. rodentium infection.

Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly.

The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.

Rhizogenic Agrobacterium protein RolB interacts with the TOPLESS repressor proteins to reprogram plant immunity and development.

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.

Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis.

The chaperone SecB has been implicated in de novo protein folding and translocation across the membrane, but it remains unclear which nascent polypeptides SecB binds, when during translation SecB acts, how SecB function is coordinated with other chaperones and targeting factors, and how polypeptide engagement contributes to protein biogenesis. Using selective ribosome profiling, we show that SecB binds many nascent cytoplasmic and translocated proteins generally late during translation and controlled by the chaperone trigger factor. Revealing an uncharted role in co-translational translocation, inner membrane proteins (IMPs) are the most prominent nascent SecB interactors. Unlike other substrates, IMPs are bound early during translation, following the membrane targeting by the signal recognition particle. SecB remains bound until translation is terminated, and contributes to membrane insertion. Our study establishes a role of SecB in the co-translational maturation of proteins from all cellular compartments and functionally implicates cytosolic chaperones in membrane protein biogenesis.

Hidden in plain sight: challenges in proteomics detection of small ORF-encoded polypeptides.

Genomic studies of bacteria have long pointed toward widespread prevalence of small open reading frames (sORFs) encoding for short proteins, <100 amino acids in length. Despite the mounting genomic evidence of their robust expression, relatively little progress has been made in their mass spectrometry-based detection and various blanket statements have been used to explain this observed discrepancy. In this study, we provide a large-scale riboproteogenomics investigation of the challenging nature of proteomic detection of such small proteins as informed by conditional translation data. A panel of physiochemical properties alongside recently developed mass spectrometry detectability metrics was interrogated to provide a comprehensive evidence-based assessment of sORF-encoded polypeptide (SEP) detectability. Moreover, a large-scale proteomics and translatomics compendium of proteins produced by Salmonella Typhimurium (S. Typhimurium), a model human pathogen, across a panel of growth conditions is presented and used in support of our in silico SEP detectability analysis. This integrative approach is used to provide a data-driven census of small proteins expressed by S. Typhimurium across growth phases and infection-relevant conditions. Taken together, our study pinpoints current limitations in proteomics-based detection of novel small proteins currently missing from bacterial genome annotations.

Small Protein Enrichment Improves Proteomics Detection of sORF Encoded Polypeptides.

With the rapid growth in the number of sequenced genomes, genome annotation efforts became almost exclusively reliant on automated pipelines. Despite their unquestionable utility, these methods have been shown to underestimate the true complexity of the studied genomes, with small open reading frames (sORFs; ORFs typically considered shorter than 300 nucleotides) and, in consequence, their protein products (sORF encoded polypeptides or SEPs) being the primary example of a poorly annotated and highly underexplored class of genomic elements. With the advent of advanced translatomics such as ribosome profiling, reannotation efforts have progressed a great deal in providing translation evidence for numerous, previously unannotated sORFs. However, proteomics validation of these riboproteogenomics discoveries remains challenging due to their short length and often highly variable physiochemical properties. In this work we evaluate and compare tailored, yet easily adaptable, protein extraction methodologies for their efficacy in the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model pathogen Salmonella typhimurium (S. typhimurium). Further, an optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 and relying on differential peptide vs. protein solubility was developed and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

Use of Hybrid Data-Dependent and -Independent Acquisition Spectral Libraries Empowers Dual-Proteome Profiling.

In the context of bacterial infections, it is imperative that physiological responses can be studied in an integrated manner, meaning a simultaneous analysis of both the host and the pathogen responses. To improve the sensitivity of detection, data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows in identifying and quantifying low-abundant proteins. Here, by making use of representative bacterial pathogen/host proteome samples, we report an optimized hybrid library generation workflow for DIA mass spectrometry relying on the use of data-dependent and in silico-predicted spectral libraries. When compared to searching DDA experiment-specific libraries only, the use of hybrid libraries significantly improved peptide detection to an extent suggesting that infection-relevant host-pathogen conditions could be profiled in sufficient depth without the need of a priori bacterial pathogen enrichment when studying the bacterial proteome. Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD017904 and PXD017945.

Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.

Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.

Ablation of elongation factor 2 kinase enhances heat-shock protein 90 chaperone expression and protects cells under proteotoxic stress.

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation stage of mRNA translation and is activated under different stress conditions to slow down protein synthesis. One effect of eEF2K is to alter the repertoire of expressed proteins, perhaps to aid survival of stressed cells. Here, we applied pulsed stable isotope labeling with amino acids in cell culture (SILAC) to study changes in the synthesis of specific proteins in human lung adenocarcinoma (A549) cells in which eEF2K had been depleted by an inducible shRNA. We discovered that levels of heat-shock protein 90 (HSP90) are increased in eEF2K-depleted human cells as well as in eEF2K-knockout (eEF2K-/-) mouse embryonic fibroblasts (MEFs). This rise in HSP90 coincided with an increase in the fraction of HSP90 mRNAs associated with translationally active polysomes, irrespective of unchanged total HSP90 levels. These results indicate that blocking eEF2K function can enhance expression of HSP90 chaperones. In eEF2K-/- mouse embryonic fibroblasts (MEFs), inhibition of HSP90 by its specific inhibitor AUY922 promoted the accumulation of ubiquitinated proteins. Notably, HSP90 inhibition promoted apoptosis of eEF2K-/- MEFs under proteostatic stress induced by the proteasome inhibitor MG132. Up-regulation of HSP90 likely protects cells from protein folding stress, arising, for example, from faster rates of polypeptide synthesis due to the lack of eEF2K. Our findings indicate that eEF2K and HSPs closely cooperate to maintain proper proteostasis and suggest that concomitant inhibition of HSP90 and eEF2K could be a strategy to decrease cancer cell survival.

eIF1 modulates the recognition of suboptimal translation initiation sites and steers gene expression via uORFs.

Alternative translation initiation mechanisms such as leaky scanning and reinitiation potentiate the polycistronic nature of human transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by the eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4000 proteins and 10 000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. Here, the stringency of start codon selection and preference for an optimal nucleotide context were largely diminished leading to translational upregulation of uORFs with suboptimal start. Interestingly, genes affected by eIF1 deprivation were implicated in energy production and sensing of metabolic stress.

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes.

Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.

A Role for Human N-alpha Acetyltransferase 30 (Naa30) in Maintaining Mitochondrial Integrity.

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.

Proteomic Identification of Cysteine Cathepsin Substrates Shed from the Surface of Cancer Cells.

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192.

N-terminal acetylome analysis reveals the specificity of Naa50 (Nat5) and suggests a kinetic competition between N-terminal acetyltransferases and methionine aminopeptidases.

Cotranslational N-terminal (Nt-) acetylation of nascent polypeptides is mediated by N-terminal acetyltransferases (NATs). The very N-terminal amino acid sequence largely determines whether or not a given protein is Nt-acetylated. Currently, there are six distinct NATs characterized, NatA-NatF, in humans of which the in vivo substrate specificity of Naa50 (Nat5)/NatE, an alternative catalytic subunit of the human NatA, so far remained elusive. In this study, we quantitatively compared the Nt-acetylomes of wild-type yeast S. cerevisiae expressing the endogenous yeast Naa50 (yNaa50), the congenic strain lacking yNaa50, and an otherwise identical strain expressing human Naa50 (hNaa50). Six canonical yeast NatA substrates were Nt-acetylated less in yeast lacking yNaa50 than in wild-type yeast. In contrast, the ectopically expressed hNaa50 resulted, predominantly, in the Nt-acetylation of N-terminal Met (iMet) starting N-termini, including iMet-Lys, iMet-Val, iMet-Ala, iMet-Tyr, iMet-Phe, iMet-Leu, iMet-Ser, and iMet-Thr N-termini. This identified hNaa50 as being similar, in its substrate specificity, to the previously characterized hNaa60/NatF. In addition, the identification, in yNaa50-lacking yeast expressing hNaa50, of Nt-acetylated iMet followed by a small residue such as Ser, Thr, Ala, or Val, revealed a kinetic competition between Naa50 and Met-aminopeptidases (MetAPs), and implied that Nt-acetylated iMet followed by a small residue cannot be removed by MetAPs, a deduction supported by our in vitro data. As such, Naa50-mediated Nt-acetylation may act to retain the iMet of proteins of otherwise MetAP susceptible N-termini and the fraction of retained and Nt-acetylated iMet (followed by a small residue) in such a setting would be expected to depend on the relative levels of ribosome-associated Naa50/NatA and MetAPs.

Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects.

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.

Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B.

BACKGROUND: Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB). RESULTS: In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3' position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1' position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5' leader sequence (5'UTR) of BNIP-2. CONCLUSIONS: Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.