Sequence B2 Research Articles

Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation.

N4-acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N-acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10-mediated ac4C in HCC progression and provide a promising therapeutic approach. The ac4C levels were evaluated by dot blot and ultra-performance liquid chromatography-tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10-ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull-down, mass spectrometry, and site-specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10-ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high-throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10-mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. Our study identified a novel oncogenic epi-transcriptome axis involving NAT10-ac4C/eEF2-HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

Symmetry Breaking with the SCAN Density Functional Describes Strong Correlation in the Singlet Carbon Dimer.

The SCAN (strongly constrained and appropriately normed) meta-generalized gradient approximation (meta-GGA), which satisfies all 17 exact constraints that a meta-GGA can satisfy, accurately describes equilibrium bonds that are normally correlated. With symmetry breaking, it also accurately describes some sd equilibrium bonds that are strongly correlated. While sp equilibrium bonds are nearly always normally correlated, the C2 singlet ground state is known from correlated wave function theory to be a rare case of strong correlation in an sp equilibrium bond. Earlier work that calculated atomization energies of the molecular sequence B2, C2, O2, and F2 in the local spin density approximation (LSDA), the Perdew-Burke-Ernzerhof (PBE) GGA, and the SCAN meta-GGA, without symmetry breaking in the molecule, found that only SCAN was accurate enough to reveal an anomalous under-binding for C2. This work shows that spin symmetry breaking in singlet C2, which involves the appearance of net up- and down-spin densities on opposite sides (not ends) of the bond, corrects that underbinding, with a small SCAN atomization-energy error more like that of the other three molecules, suggesting that symmetry breaking with an advanced density functional might reliably describe strong correlation. This article also discusses some general aspects of symmetry breaking and the insights into strong correlation that symmetry breaking can bring. The normally correlated low-lying triplet excited state has the right vertical excitation energy in SCAN but not in LSDA or PBE, where the triplet is a false ground state. Fractional occupation numbers are found only for the symmetry-unbroken singlet and only in LSDA and PBE GGA.

Genome Sequence of Eubacterium limosum B2 and Evolution for Growth on a Mineral Medium with Methanol and CO2 as Sole Carbon Sources

Eubacterium limosum is an acetogen that can produce butyrate along with acetate as the main fermentation end-product from methanol, a promising C1 feedstock. Although physiological characterization of E. limosum B2 during methylotrophy was previously performed, the strain was cultured in a semi-defined medium, limiting the scope for further metabolic insights. Here, we sequenced the complete genome of the native strain and performed adaptive laboratory evolution to sustain growth on methanol mineral medium. The evolved population significantly improved its maximal growth rate by 3.45-fold. Furthermore, three clones from the evolved population were isolated on methanol mineral medium without cysteine by the addition of sodium thiosulfate. To identify mutations related to growth improvement, the whole genomes of wild-type E. limosum B2, the 10th, 25th, 50th, and 75th generations, and the three clones were sequenced. We explored the total proteomes of the native and the best evolved clone (n°2) and noticed significant differences in proteins involved in gluconeogenesis, anaplerotic reactions, and sulphate metabolism. Furthermore, a homologous recombination was found in subunit S of the type I restriction-modification system between both strains, changing the structure of the subunit, its sequence recognition and the methylome of the evolved clone. Taken together, the genomic, proteomic and methylomic data suggest a possible epigenetic mechanism of metabolic regulation.

Sequence stratigraphy of the ca 1640 Ma Barney Creek Formation, McArthur Basin, Australia

The ca 1640 Ma Barney Creek Formation is a mostly fine-grained silicicastic unit of the McArthur Group in the southern McArthur Basin of northern Australia. It is one of the most prospective units for clastic-dominated (CD-type) Zn–Pb deposits in the world, hosting the giant McArthur River Zn–Pb–Ag and the Teena Zn–Pb deposits. The Barney Creek Formation records deposition of dominantly dolomitic siltstone turbidite facies at or below storm wave base. These strata were deposited in a series of sub-basins and on submerged paleohighs that formed in response to roughly north–south extension during early Barney Creek times. A detailed, regional-scale sequence stratigraphic analysis based on 20 wells indicates that the middle McArthur Group (Emmerugga Dolostone to Lynott Formation) comprises six third-order depositional sequences, of which the Barney Creek Formation includes two (named B1 and B2). These two sequences and the overlying sequence L1 (Reward Dolostone and lower Lynott Formation) record significant lateral facies and thickness changes across the southern McArthur Basin. These variations can be explained by a geographical shift in the depocentre during structural re-organisation of the basin. The depocentre migrated from the south, where accommodation was mostly created during deposition of sequence B1, towards the north during deposition of sequence B2. KEY POINTS Sequence stratigraphic analysis of the Barney Creek Formation on a regional scale based on 20 wells. Barney Creek Formation includes two third-order depositional sequences (named B1 and B2), which record significant lateral thickness and facies shifts. Thickness and facies shifts can be explained by geographical shift of depocentre from the present-day south to north.

Structure and Multistage Martensite Transformation in Nanocrystalline Ti-50.9Ni Alloy

An electron microscopic study of the evolution of the size, morphology, and spatial distribution of coherent Ti3Ni4 particles with a change in the aging temperature in a nanocrystalline (NC) Ti-50.9 at % Ni alloy with an inhomogeneous grain–subgrain B2-austenitic nanostructure has been carried out. It was found that with an increase in the aging temperature, along with a change in the size and shape of Ti3Ni4 nanoparticles, their spatial distribution changes from location at dislocations to precipitates at subboundaries. Research has shown that the presence of different types of internal interfaces in the nanostructure contributes to the heterogeneous distribution of coherent Ti3Ni4 nanoparticles in the volume of the B2 matrix, which is associated with the precipitation of particles in the region of low-angle subboundaries and the suppression of the Ti3Ni4 precipitation in nanograins with high-angle boundaries. The difference in the structural-phase state of nanograins and subgrains regions is the main reason for the implementation of the anomalous R-phase transformation effect in the sequence of multistage martensitic transformations B2↔R↔B19′.

Discovery of a Novel Long Noncoding RNA Lx8-SINE B2 as a Marker of Pluripotency.

Pluripotency and self-renewal of embryonic stem cells (ESCs) are marked by core transcription regulators such as Oct4, Sox2, and Nanog. Another important marker of pluripotency is the long noncoding RNA (lncRNA). Here, we ind that a novel long noncoding RNA (lncRNA) Lx8-SINE B2 is a marker of pluripotency. LncRNA Lx8-SINE B2 is enriched in ESCs and downregulated during ESC differentiation. By rapid amplification of cDNA ends, we identified the full-length sequence of lncRNA Lx8-SINE B2. We further showed that transposable elements at upstream of lncRNA Lx8-SINE B2 could drive the expression of lncRNA Lx8-SINE B2. Furthermore, ESC-specific expression of lncRNA Lx8-SINE B2 was driven by Oct4 and Sox2. In summary, we identified a novel marker lncRNA of ESCs, which is driven by core pluripotency regulators.

Ti3Ni4 precipitation features in heat-treated grain / subgrain nanostructure in Ni-rich TiNi alloy

The effect of the annealing temperature of a Ti-50.9 at.% Ni nanocrystalline alloy with an inhomogeneous grain/subgrain structure on Ti3Ni4 precipitation was investigated. The evolution of sizes, morphology, and spatial distribution of Ti3Ni4 particles after annealing at 300 °C, 400 °C and 500 °C studied. Found that Ti3Ni4 particles nucleate by a heterogeneous mechanism predominantly in the substructure and almost not observed in nanograins. With an increase in the temperature of annealing the Ti3Ni4 particles increase in size and their spatial distribution changes from location in subgrains at dislocations (at 300 °C) to precipitation at subboundaries (at 400 °C). The features of recovery and recrystallization in the grain/subgrain structure at 500 °C identified and their relationship with the processes of dissolution and coagulation of Ti3Ni4 particles revealed. The large-scale microstructural heterogeneity leading to different precipitation behavior between substructure regions and nanograins groups is considered as an additional cause of the multi-stage martensite transformation sequence B2 ↔ R ↔ B19′.

Genome Sequence of a Potentially New Buttiauxella Species, Strain B2, Isolated from Rhizosphere of Olivillo Trees (Aextoxicon punctatum).

We announce the draft genome sequence of strain B2, which belongs to a potentially new Buttiauxella species, isolated from soil associated with rhizosphere of olivillo trees (Aextoxicon punctatum). Its size is 4,967,099 bp, and its G+C content is 49.1%. The genome of strain B2 carries genes related to rhizobacteria that promote the growth of plants.

Мартенситные превращения сплавов на основе никелида титана с разными легирующими добавками

Investigations of the martensitic transformations of TiNi-based alloys grade TN-10, doped with 1 at.% V, Nb, Ag, were presented. It was shown that alloying elements (Ag, Nb, V) cause an increase in internal elastic stresses inside the crystalline structure of the B2 phase, which leads to an increase in the beginning temperature of direct martensitic transformation. Of the selected alloying elements, vanadium has a significant effect on this temperature, shifting it to the region of high values ​​by ~ 45 ° C. In alloys with vanadium and silver, a complex sequence of martensitic transformations B2-> R-> B19 'is realized, and in alloys with niobium, a simple B2-> B19' is realized.

Synthesis and Characterization of Kalata B2 Cyclotide (GLPVCGETCFGGTCNTPGCSCTWPICTRD) on Wang Resin, as Solid Support

With the help of the solid phase peptide synthesizing method, we synthesized Kalata B2 cyclotide and the final product was purified by preparative HPLC. The sequence of Kalata B2 is GLPVCGETCFGGTCNTPGCSCTWPICTRD and its m/z M + H mass is 2955.53. It had been first isolated from Oldenlandia affinis plant leaves extract which belongs to Rubiaceae family. The synthesis of Kalata B2 has been characterized through UPLC/MS, LC/MS, CD, 1HNMR, and IR.

In Situ Structural Characterization of Functionally Graded Ni–Ti Shape Memory Alloy During Tensile Loading

A functionally graded NiTi shape memory alloy wire was investigated by in situ synchrotron radiation-based X-ray diffraction (SR-XRD) during cyclic tensile deformation. The transformation temperatures were determined by DSC and the thermomechanical behaviour was analysed by three-point bending test. The present study focussed on the localized heat treatment (Joule heat effect, reaching 300 °C, 350 and 400 °C pulses for 10 min) of NiTi wires, using an equipment that allows a large variety of graded conditions. Structural, mechanical and thermomechanical characterization is presented to get a perspective of the different types of graded functionality. A combination of two strategies has been used for the in situ analysis by SR-XRD of the tensile tests: (i) continuously following the structural evolution at one single point (at the center of the heat-treated segment) all long the load/unload cycle and (ii) scanning the full heat-treated segmentat previously defined discrete steps of the stress–strain curve. The combined information from both types of tests provided detailed information about the phase transformations taking place in different regions of the functionally graded segment, at different steps of the tensile load/unload cycle, giving a better understanding of the overall mechanical, namely the evidence of the sequence B2 ↔ R ↔ B19′ for the direct and reverse transformations.

Evolution of the Urgonian shallow-water carbonate platform on the Helvetic shelf during the late Early Cretaceous

Urgonian platform carbonate (Cretaceous; Barremian, Aptian) forms an important lithostratigraphic unit in the Helvetic fold- and thrust unit of the northern Swiss Alps. Its widespread distribution and ubiquity allow for an integrated high-resolution study of macro- and microfacies, benthic foraminiferal biostratigraphy, sequence stratigraphy, and carbon-isotope and phosphorus records. The resulting data confirm the importance of environmental forcing and sea level change on the style of carbonate production and accumulation along the margin of the northern Tethys during the late Early Cretaceous. Stratal geometries, the succession of microfacies, the identification of major emersion surfaces, and biostratigraphy observed and analysed in twelve sections through the inner, middle, and outer platform, and in the panorama of the Churfirsten range, permit subdivision of the analysed succession into eight depositional sequences. The succession starts with a phase of sedimentary condensation (Altmann Member (Mb); late Hauterivian – late early Barremian; sequences H7, H8, and B1), followed by the deposition of hemipelagic sediments (Drusberg Mb; restricted to sequence B2 of the late early Barremian on the inner platform; and covering the late early Barremian to the middle late Barremian on the outer platform, and to the early Aptian on the outer shelf, thereby showing an important diachroneity of its upper boundary related to the inception and progradation of the Urgonian platform), and the development of predominantly lagoonal carbonate (Schrattenkalk Formation (Fm); early late Barremian – early Aptian; sequences B3, B4, B5, and A1). The Schrattenkalk Fm documents important progradation and aggradation of the carbonate platform, and the change from a ramp-like to flat-topped geometry. The oldest, allochthonous remains of the shallow-water carbonate platform were identified intercalated in and on top of the lower Barremian Drusberg Mb (sequence B2). Sequence boundary (SB) B3 resulted from an important regressive phase near the early-late Barremian boundary, which led to the emersion of the hemipelagic sediments of the Drusberg Mb in the inner part of the shelf and to the deposition of a lowstand systems tract at the base of the Lower Schrattenkalk Mb (late Barremian; sequences B3–5) in intermediate and distal domains. Deposition of in-situ platform carbonate started during the following transgressive phase in the middle late Barremian, which flooded the entire investigated area. The associated faunal assemblages and phosphorus contents indicate a concomitant increase in nutrient input, which led to a mixed photozoan-heterozoan platform association dominated by annelids and flat orbitolinids, and the formation of a condensed phosphate-rich bed on the outer shelf (Chopf Bed; middle late Barremian). The subsequent sea-level highstand allowed for the deposition of the first typical Urgonian carbonates rich in corals and rudists. This depositional sequence (B3) terminated by the important infilling of accommodation space combined with sea-level fall of at least 15 m. Later on, close to the Barremian-Aptian boundary, a further, major emersion phase (SB A1) was triggered by sea-level fall, estimated here as at least 16 m, which terminated this first phase in the deposition of rudist and coral-rich platform carbonates covering the middle late to latest Barremian (B3–B5). The overlying Rawil Mb (lowermost Aptian; transgressive systems tract A1) resulted from progressive deepening and document a phase of increasing eutrophication of the depositional environment, resulting in a mixed siliciclastic-carbonate platform build-up, characterized by sea-grass facies and the massive occurrence of Palorbitolina lenticularis. The overlying Upper Schrattenkalk Mb (lower Aptian; highstand systems tract A1) records recovery of the rudist-rich photozoan Urgonian platform. Its subsequent demise occurred well before the Selli oceanic anoxic episode (OAE 1a). It was initiated by emersion of the platform due to high-amplitude sea-level fall (SB A2), followed by eutrophication during the subsequent transgressive phase.The carbon-isotope records show an increase towards more positive values during the Lower Schrattenkalk Mb and the base of the Rawil Mb, interrupted in most sections by an excursion to lower values near the Barremian–Aptian boundary. A shift to lower values occurred also in the uppermost part of the Rawil Mb, followed by variable trends in the Upper Schrattenkalk Mb. These long-term trends are well correlated with the basinal record (Angles, La Bédoule). Deviations in the correlations are related to the influence of facies and microfacies, primary mineralogy, emersion phases, and post-depositional alteration.

Draft Genome Sequence of Clostridium botulinum B2 450 Strain from Wound Botulism in a Drug User in Italy.

Here, we report the draft genome sequence of Clostridium botulinum B2 450, responsible for the first reported case of wound botulism in a drug user in Italy.

Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin β chain short arm regions

Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1–α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255–266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604–616), B2–105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081–1092), and B3–19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182–193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2–105, and B3–19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3–19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2–64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618–630), the Glu8 residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins.

Quantitative microstructure analyses upon multistage martensitic transformation in an aged Ti–50.8at.% Ni alloy

Abstract Quantitative microstructure analyses upon multistage martensitic transformation (MMT) in an aged Ti–50.8 at.% Ni alloy have been performed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), electron back scattered diffraction (EBSD) and transmission electron microscopy (TEM). The alloy used was heat-treated at 1273 K for 3.6 ks and then aged at 773 K for 3.6 ks under regulated and unregulated atmospheres. The specimen heat-treated under the regulated atmosphere shows the single stage martensitic transformation in addition to the R-phase transformation. That is, B2 → R → M. On the other hand, the specimen heat-treated under the unregulated atmosphere shows the MMT with the sequence of B2 → R → M1 → M2 → M3. There are three exothermic peaks related to the martensitic transformation in addition to the R-phase transformation peak. The R-phase transformation takes place at the intermediate part of grains and around the grain boundaries. Subsequently, the first (M1) and second (M2) martensitic transformations occur at the intermediate part of grains and around the grain boundaries, respectively. Finally, the third transformation (M3) appears at the center part of grains directly from the B2 phase without via the R-phase transformation. The proposed transformation sequence is quantitatively proved by comparing the EBSD image with DSC curve.

The Jurassic–Cretaceous basaltic magmatism of the Oued El-Abid syncline (High Atlas, Morocco): Physical volcanology, geochemistry and geodynamic implications

Basaltic lava flows, dykes and sills, interbedded within red clastic continental sedimentary sequences (the so called “Couches Rouges”) are widespread in the Oued El-Abid syncline. They represent the best candidates to study the Jurassic–Cretaceous magmatism in the Moroccan High Atlas. The volcanic successions were formed during two pulses of volcanic activity, represented by the Middle to Upper Jurassic basaltic sequence B1 (1–4 eruptions) and the Lower Cretaceous basaltic sequence B2 (three eruptions). Whether belonging to the B1 or B2, the lava flows present morphology and internal structures typical of inflated pahoehoe. Our geochemical data show that, at least for Jurassic magmatism, the dykes, and sills cannot be considered as strictly representing the feeders of the sampled lava flows. The Middle to Upper Jurassic pulse is moderately alkaline in character, while the Lower Cretaceous one is transitional. Crustal contamination plays a minor role in the petrogenesis of these magmas, which were generated by variable partial melting degrees of a garnet-bearing mantle source. Magmatism location was controlled by pre-existing Hercynian fault systems reactivated during a Middle to Upper Jurassic–Cretaceous rifting event. The associated lithospheric stretching induced melting, by adiabatic decompression, of enriched low-solidus infra-lithospheric domains.

Genome Sequence of the Human-Pathogenetic Bacterium Vibrio vulnificus B2

Vibrio vulnificus, which can lead to rapidly expanding cellulitis or septicemia, is present in the marine environment. Here, we present the draft genome sequence of strain B2, which was isolated from a septicemia patient in 2010.

Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.

Molecular Characterization of Bubaline Integrin β2 (ITGB2) cDNA

Sharma, D., Niranjan, S.K., Kumar, S., Deb, S.M., Naskar, S., Sharma, A. and Mitra, A. 2010. Molecular characterization of bubaline integrin β2 (ITGB2) cDNA. J. Appl. Anim. Res., 37: 217–220. A 2310-bp long buffalo (Bubalus bubalis) cDNA sequence of Integrin b2 (ITGB2) gene was characterized. At nucleotide sequence level, buffalo ITGB2 exhibited 96–98% homologies with other ruminants like cattle, bison, sheep, goat and deer. In coding region, total thirty two nucleotide differences have been observed between buffalo and cattle sequences including 15 non-homologous substitutions. Importantly, a conserved site (22) in PSI domain of the ruminants (Thr) was substituted in buffalo (Ala) indicating normal function of the integrin unless the second mutation in CD lie molecule coexists. Buffalo ITGB2 has seven differences in I-like domain region as compared to cattle. The transmembrane and cytoplasmic regions were highly conserved among buffalo and other ruminants. High homology and close phylogenetic association among ITGB2 sequences of the ruminants including buffalo indicates highly conserved nature of the gene.

ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.