The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with ...the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull‐down experiments using the proteasome regulatory complex (proteasome‐activating nucleotidase PAN) as bait. X‐ray crystallography and small‐angle X‐ray scattering experiments revealed that the protein is monomeric and adopts a β‐barrel core structure with an oligonucleotide/oligosaccharide‐binding (OB)‐fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)‐binding properties. Pull‐downs, co‐immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull‐downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN–20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome‐Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA‐processing enzymes and exosome subunits dependent on Pbp11's N‐terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.
Partner of the archaeal proteasome PAN:20S complex in Thermococcales, Pbp11 directly interacts with the unfoldase PAN. From the cellular extract, Pbp11 pulls down the proteasome system and other macromolecular assemblies related to RNA processes. These last interactions are dependent on the presence of the flexible N‐terminal tail of Pbp11, a key feature of Pbp11 to bind transfer ribonucleic acids. Pbp11 becomes an interesting candidate to study tight connections between these nanomachines in the context of extremophilic Archaea.
Matrix metalloproteinases outside vertebrates Marino-Puertas, Laura; Goulas, Theodoros; Gomis-Rüth, F. Xavier
Biochimica et biophysica acta. Molecular cell research,
November 2017, 2017-11-00, 20171101, Letnik:
1864, Številka:
11
Journal Article
Recenzirano
Odprti dostop
The matrix metalloproteinase (MMP) family belongs to the metzincin clan of zinc-dependent metallopeptidases. Due to their enormous implications in physiology and disease, MMPs have mainly been ...studied in vertebrates. They are engaged in extracellular protein processing and degradation, and present extensive paralogy, with 23 forms in humans. One characteristic of MMPs is a ~165-residue catalytic domain (CD), which has been structurally studied for 14 MMPs from human, mouse, rat, pig and the oral-microbiome bacterium Tannerella forsythia. These studies revealed close overall coincidence and characteristic structural features, which distinguish MMPs from other metzincins and give rise to a sequence pattern for their identification. Here, we reviewed the literature available on MMPs outside vertebrates and performed database searches for potential MMP CDs in invertebrates, plants, fungi, viruses, protists, archaea and bacteria. These and previous results revealed that MMPs are widely present in several copies in Eumetazoa and higher plants (Tracheophyta), but have just token presence in eukaryotic algae. A few dozen sequences were found in Ascomycota (within fungi) and in double-stranded DNA viruses infecting invertebrates (within viruses). In contrast, a few hundred sequences were found in archaea and >1000 in bacteria, with several copies for some species. Most of the archaeal and bacterial phyla containing potential MMPs are present in human oral and gut microbiomes. Overall, MMP-like sequences are present across all kingdoms of life, but their asymmetric distribution contradicts the vertical descent model from a eubacterial or archaeal ancestor. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.
•Matrix metalloproteinase (MMP) have been studied in vertebrates, where they participate in extracellular protein processing.•Current searches identified potential MMPs in invertebrates, plants, fungi, viruses, protists, archaea and bacteria.•MMP-like sequences are present across all kingdoms of life, but their distribution contradicts the vertical descent model.
Peptidases must be exquisitely regulated to prevent erroneous cleavage and one control is provided by protein inhibitors. These are usually specific for particular peptidases or families and ...sterically block the active-site cleft of target enzymes using lock-and-key mechanisms. In contrast, members of the +1400-residue multi-domain α
-macroglobulin inhibitor family (α
Ms) are directed against a broad spectrum of endopeptidases of disparate specificities and catalytic types, and they inhibit their targets without disturbing their active sites. This is achieved by irreversible trap mechanisms resulting from large conformational rearrangement upon cleavage in a promiscuous bait region through the prey endopeptidase. After decades of research, high-resolution structural details of these mechanisms have begun to emerge for tetrameric and monomeric α
Ms, which use ‘Venus-flytrap’ and ‘snap-trap’ mechanisms, respectively. In the former, represented by archetypal human α
M, inhibition is exerted through physical entrapment in a large cage, in which preys are still active against small substrates and inhibitors that can enter the cage through several apertures. In the latter, represented by a bacterial α
M from
, covalent linkage and steric hindrance of the prey inhibit activity, but only against very large substrates.
The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with ...the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Transforming growth factor β is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFβ1-TGFβ3) engaged in signaling functions through binding of cognate TGFβ ...receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFβs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFβs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFβ2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFβ2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFβ receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGβ2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.
Matrix metalloproteinases (MMPs) occur in 23 human paralogues with key functions in physiology, and their activity is controlled by protein inhibitors. Reversion-inducing cysteine-rich protein with ...Kazal motifs (RECK), which is essential for embryogenesis and tumour suppression, has been reported to inhibit MMPs. Here, we developed eukaryotic and bacterial expression systems for different RECK variants and analysed their inhibitory capacity against representative MMPs in vitro. We could not detect any significant inhibition. Instead, we found that partially purified RECK from the conditioned medium of transfected Expi293F cells but not that of ExpiCHO-S or Drosophila Schneider cells contained a contaminant with proteolytic activity. The contaminant was removed through treatment with a small-molecule serine peptidase inhibitor and additional chromatographic purification. A tantamount contaminant was further detected in an equivalent expression system of the N-terminal fragment of the proteoglycan testican 3, but not in those of two other proteins. These results indicate that previous reports of inhibitory activity of recombinant RECK on MMPs, which were performed with partially purified samples, were probably masked by a coeluting contaminant present in the supernatant of HEK293-derived cells. Thus, RECK is probably not a direct inhibitor of MMP catalytic activity but may still regulate MMPs through other mechanisms.
Abstract
α
2
-Macroglobulins (α
2
Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α
2
Ms have ...been studied over decades using authentic material from primary sources, which was limited by sample heterogeneity and contaminants. Here, we developed high-yield expression systems based on transient transfection in
Drosophila
Schneider 2 and human Expi293F cells, which produced pure human α
2
M (hα
2
M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα
2
M was mainly found in the induced form. Shorter hα
2
M variants encompassing N-/C-terminal parts were also expressed and yielded pure material at ~1.6/~1.3 and ~3.2/~4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic hα
2
M to recombinant latent human transforming growth factor-β
2
(pro-TGF-β
2
) and bacterial G-related α
2
M binding protein (GRAB) by surface plasmon resonance, multiple-angle laser light scattering, size-exclusion chromatography, fluorogenic labelling, gel electrophoresis and Western-blot analysis. Two GRAB molecules formed stable complexes of high affinity with native and induced authentic hα
2
M tetramers. The shorter recombinant hα
2
M variants interacted after preincubation only. In contrast, pro-TGF-β
2
did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.
α
-Macroglobulins (α
Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α
Ms have been studied ...over decades using authentic material from primary sources, which was limited by sample heterogeneity and contaminants. Here, we developed high-yield expression systems based on transient transfection in Drosophila Schneider 2 and human Expi293F cells, which produced pure human α
M (hα
M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα
M was mainly found in the induced form. Shorter hα
M variants encompassing N-/C-terminal parts were also expressed and yielded pure material at ~1.6/~1.3 and ~3.2/~4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic hα
M to recombinant latent human transforming growth factor-β
(pro-TGF-β
) and bacterial G-related α
M binding protein (GRAB) by surface plasmon resonance, multiple-angle laser light scattering, size-exclusion chromatography, fluorogenic labelling, gel electrophoresis and Western-blot analysis. Two GRAB molecules formed stable complexes of high affinity with native and induced authentic hα
M tetramers. The shorter recombinant hα
M variants interacted after preincubation only. In contrast, pro-TGF-β
did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.
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