Prevalence of Enhanced Granular Expression of Thrombospondin Type-1 Domain-Containing 7A in the Glomeruli of Japanese Patients with Idiopathic Membranous Nephropathy

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults. Autoantibodies against M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A), which mainly belong to the IgG4 subclass, were reported as associated antibodies for the development of MN. Although PLA2R is a major target antigen for idiopathic MN, the prevalence of MN patients seropositive for PLA2R in Japan is lower than that in other countries. In this study, we conducted immunohistochemical analysis of the presence of THSD7A and PLA2R in renal specimens of MN patients to estimate the prevalence of THSD7A/PLA2R-related idiopathic MN in Japan. Enhanced granular expression of THSD7A and PLA2R was detected in 9.1% and 52.7%, respectively, of the patients with idiopathic MN. Although none of patients with secondary MN displayed enhanced granular expression of THSD7A, 5.4% of them had enhanced granular expression of PLA2R. In conclusion, the prevalence of enhanced granular expression of THSD7A in the glomeruli of Japanese patients with idiopathic MN was higher than the prevalence of MN patients seropositive for THSD7A in USA and Europe.     

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

Background

Scaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.

Methodology/Principal Findings

We have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.

Conclusions/Significance

Our results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.     

Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan

Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the ³⁶WDAWGPWSECSRTC⁴⁹ sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp³⁹ and Trp⁴². C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-³H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.The ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type-1 repeats) superfamily (1) consists of 19 secreted metalloproteases (ADAMTS proteases) and six ADAMTS-like proteins in humans. ADAMTS-like proteins closely resemble the ancillary domains of ADAMTS proteases and like them have a conserved modular organization containing one or more thrombospondin type-1 repeats (TSRs)² (2–5). TSRs are modules of ∼50 amino acids having a characteristic six-cysteine signature. The prototypic ADAMTSL, ADAMTSL1, also referred to as punctin-1 because of its punctate distribution in the substratum of transfected cells, is a 525-residue glycoprotein containing four TSRs (4). A longer punctin-1 variant arising from alternative splicing, containing 13 TSRs and homologous to ADAMTSL3, is predicted by the human genome sequencing project ({"type":"entrez-nucleotide","attrs":{"text":"NM_001040272","term_id":"334688819","term_text":"NM_001040272"}}NM_001040272) but has not yet been physically cloned and expressed. The function of ADAMTSL1/punctin-1 is unknown. Recently, ADAMTSL2 and ADAMTSL4 mutations were identified in the genetic disorders geleophysic dysplasia (6) and recessive isolated ectopia lentis, respectively (2). In genome-wide analysis, the ADAMTSL3 locus has been associated with variation in human height (7). Thus, in addition to known genetic disorders caused by ADAMTS mutations (8, 9), ADAMTSL family members are now also implicated in human disease. Among the ADAMTS proteases, ADAMTS5 and ADAMTS4 are strongly associated with cartilage destruction in arthritis (10–12).Like most secreted proteins, the ADAMTS superfamily members undergo post-translational modification and are predicted to contain N-linked oligosaccharides. In addition, TSRs of ADAMTS superfamily members, by virtue of high sequence similarity to the corresponding motifs in thrombospondin 1 and properdin, are predicted to contain two uncommon types of glycosylation. Specifically, TSRs often contain the sequence motifs W⁰XXW⁺³ and C¹X_2–3(S/T)C²XXG, which are consensus sites for protein C-mannosylation of the W⁰ residue and O-fucosylation (of Ser/Thr) respectively, in close proximity to each other (13, 14). In recently published work, it was shown that ADAMTSL1 and ADAMTS13 are modified by O-fucosylation, the covalent attachment to Ser or Thr residues of fucose or a fucose-glucose disaccharide (15, 16). Punctin-1 contains consensus sequences for O-fucosylation in all four of its TSRs, but the presence of the glycans was previously only confirmed on TSR2, -3, and -4 (16). Addition of O-fucose is mediated by protein O-fucosyltransferase 2 (POFUT2), which is a distinct transferase from that which catalyzes addition of O-linked fucose to epidermal growth factor-like repeats (POFUT1) (17, 18). A β3-glucosyltransferase subsequently adds glucose to the 3′-OH of the fucose (19, 20). It was further demonstrated that O-fucosylation, which occurs after completion of TSR folding, was rate-limiting for secretion of punctin-1 and ADAMTS13 (15, 16). This role was inferred from the following two experimental observations. 1) Expression of wild-type punctin-1 and ADAMTS13 in Lec13 cells, which do not fucosylate proteins, led to their decreased secretion (15, 16). 2) Mutation of the modified Ser or Thr residues greatly reduced secretion of punctin-1 and ADAMTS13 (15, 16).Protein C-mannosylation is the attachment of an α-mannopyranosyl residue to the indole C-2 of tryptophan via a C-C linkage (14, 21). Unlike O-fucosylation, it can utilize protein primary structure rather than tertiary structure as the determinant, i.e. mannose is added to unfolded polypeptides or unstructured synthetic peptides (22). C-Mannosylation uses dolichyl-phosphate mannose (Dol-P-Man) as the precursor and appears to be enzyme-catalyzed within the endoplasmic reticulum (23), but the responsible mannosyltransferase has not yet been identified. A variety of mammalian cell lines can perform this modification (24). Proteins known to be C-mannosylated include human RNase 2, interleukin 12, the mucins MUC5AC and MUC5B, and several proteins containing TSRs, such as thrombospondin-1, F-spondin, and components of complement (C6 and C7) and properdin (13, 21, 25–27).Krieg et al. (22) proposed a model in which the C-mannosyltransferase bound directly to the WXXW⁺³ motif, analogous to the Asn-X-(Thr/Ser) motif for N-glycosylation, and later analysis showed that both the Trp residues in the W⁰XXW⁺³XXX motif and the sole Trp residue in a (F/Y¹)XXW⁺³ motif could be modified (13). Based on meta-analysis of the C-mannosylation literature, Julenius (28) used a neural network approach to develop a prediction algorithm for protein C-mannosylation, termed NetCGlyc. This analysis suggested that Cys was an acceptable substitute for Trp at the +3 position (i.e. permitting C-mannosylation of W⁰ in a W⁰SSC motif). Julenius (28) reported a clear preference for small and/or polar residues (Ser, Ala, Gly, and Thr) at the +1 position, whereas Phe and Leu were not allowed. The NetCGlyc algorithm provides a useful guide for prediction of C-mannosylation sites, especially in the ADAMTS superfamily, which has a large number of TSRs (27). Here we specifically inquired whether the short form of punctin-1, the prototypic ADAMTSL, is modified by C-mannosylation, analyzed the role of Trp residues in the punctin TSRs, and investigated its possible functional significance in punctin-1 biosynthesis. By demonstrating that TSR1 of ADAMTS5 is also C-mannosylated, we extended the analysis to identify this unusual modification in an ADAMTS protease.

TABLE 1

Predicted C-mannosylation sites^a in the ADAMTS superfamilyOpen in a separate window^aThe full-length human reference ADAMTS sequences from GenBank™ were analyzed at the NetCGly 1.0 server for prediction of C-mannosylation sites. For prediction of O-fucosylation sites in the same peptide, the consensus sequence C¹X_2–3(S/T)C² XXG was utilized.^bThe sequence context in which the predicted modified Trp residue occurs is provided, and the residue with predicted modification is numbered. Ser/Thr residues predicted to be O-fucosylated based on the consensus sequence CXX(S/T)C are underlined.^cSequences containing predicted C-mannosylation sites that are not within TSRs are shown in italics.     

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

Background

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.

Methods

The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1⁺NIL were also compared to identify pathogen-responsive proteins.

Results

Eight proteins were either induced or upregulated in inoculated Fhb1⁺NIL when compared with mock-inoculated Fhb1⁺NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1⁺NIL when compared with Fusarium-inoculated Fhb1⁻NIL. Proteins that were differentially expressed in the Fhb1⁺NIL, not in the Fhb1⁻NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.

Conclusions

Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1⁺NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.     

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.     

Thrombospondin is Sequentially Expressed and then De-expressed During Early Pregnancy in the Rat Uterus

The expression of thrombospondin on Day 1, Day 3 and Day 6 of pregnancy has been examined in the rat, using light microscopic immunoperoxidase and electron immunogold techniques. The glycoprotein was expressed in the apical, lateral and basal uterine epithelium on Days 1 and 3 but was then de-expressed at the time of implantation on Day 6. We propose that these data suggest a role for thrombospondin in remodelling the uterine epithelium during the plasma membrane transformation, but that it does not play a part in attachment and implantation.     

Suppression of Head Regeneration by Accelerated Wound Healing in Hydra

Published evidence suggests that tissue injury is important for head regeneration in hydra [MacWilliams, 1982, 1983a,b; Kobatake and Sugiyama, 1989]. To investigate this problem in more detail, two experimental manipulations, decapitation and mirror-image grafting, were carried out. In the latter, two decapitated polyps were axially grafted to each other to make the wound openings of the two polyps juxtaposed on each other. In normal regenerates, the wound opening closed and healed in 4 to 5 hr, while in mirror-image grafts it healed in about 1 hr. The percentage of head regeneration was lower in mirror-image grafts than that after decapitation. The effect of mirror-image grafting on morphogenetic potential levels was examined using a lateral transplantation technique. Head inhibition levels dropped in both types of regenerates to a similar extent. Head activation levels rose more in normal regenerates than in mirror-image grafts. These results show clearly that the drop in head inhibition level is due to removal of the head and is not affected by grafting. They also show that the increase in head activation levels and in the percentage of head regeneration is affected substantially by the grafting. These observations are consistent with the view that decapitation produced a greater injury effect than mirror-image grafting, and this injury effect raised the head activation level whereas it did not alter the head inhibition level. The fact that the wound remained open for a longer time in normal regenerates than in the grafts suggests that the injury effect depends not on tissue injury itself but on the length of time the wound is open.     

毕赤酵母表达蛋白质的糖基化

毕赤酵母表达系统可对表达产物进行翻译后加工如糖基化等。通过研究其糖基化的特点、影响因素以及对重组蛋白质质产量、功能的影响,介绍如何避免过度糖基化以使表达产物更加符合人类需求。     

Expression of CD1a and Type-1 Polarization Are Dissociated in Human Monocyte-Derived Dendritic Cells

Ex vivo generated monocyte-derived dendritic cell (moDC)-vaccines have long been touted as promising immunotherapeutic agents for cancer treatment, although the response rate generally remains low. The reasons for this are still unclear and confounded by the diversity in manufacturing protocols that may affect moDC function. Preclinical studies have shown that the stimulatory function of dendritic cells can be improved by engaging invariant NKT cells in vivo through the presentation of the glycolipid alpha-galactosylceramide via CD1d. However, expression of CD1d on moDC has been shown to be negatively correlated with expression of CD1a, which in turn has been suggested to be a surrogate marker for IL-12 secreting type-1 polarized moDC, the preferred functional characteristics for cancer vaccines. Here we challenge this notion by showing that plasma-derived lipids drive functional levels of CD1d expression, while CD1a expression can vary considerably in these cells without being correlated with a loss of polarization or immunogenicity.     

Presence of a Simple Tandem Repeat in the ITS1 Region of the Xylariales

A Simple Tandem Repeat sequence of 11 nucleotides has been found in the ITS1 region of the rDNA of members of Order Xylariales. The number of repetitions detected ranged from one to six, and they could be found in pure tandem or interspersed. The same core sequences have also been found in DNA from other organisms, although usually not repeated in tandem. These repetitions could have been generated by slipped strand mispairing. The presence of this sequence increases the normal rate of divergence in the ITS1 of the Xylariales. The phylogenetic implications of the presence of this sequence in the molecular taxonomy of Xylariales are also discussed. Received: 19 October 2000 / Accepted: 21 December 2000     

ZNF385B and VEGFA Are Strongly Differentially Expressed in Serous Ovarian Carcinomas and Correlate with Survival

Background

The oncogenesis of ovarian cancer is poorly understood. The aim of this study was to identify mRNAs differentially expressed between moderately and poorly differentiated (MD/PD) serous ovarian carcinomas (SC), serous ovarian borderline tumours (SBOT) and superficial scrapings from normal ovaries (SNO), and to correlate these mRNAs with clinical parameters including survival.

Methods

Differences in mRNA expression between MD/PD SC, SBOT and SNO were analyzed by global gene expression profiling (n = 23), validated by RT-qPCR (n = 41) and correlated with clinical parameters.

Results

Thirty mRNAs differentially expressed between MD/PD SC, SBOT and SNO were selected from the global gene expression analyses, and 21 were verified (p<0.01) by RT-qPCR. Of these, 13 mRNAs were differentially expressed in MD/PD SC compared with SNO (p<0.01) and were correlated with clinical parameters. ZNF385B was downregulated (FC = −130.5, p = 1.2×10⁻⁷) and correlated with overall survival (p = 0.03). VEGFA was upregulated (FC = 6.1, p = 6.0×10⁻⁶) and correlated with progression-free survival (p = 0.037). Increased levels of TPX2 and FOXM1 mRNAs (FC = 28.5, p = 2.7×10⁻¹⁰ and FC = 46.2, p = 5.6×10⁻⁴, respectively) correlated with normalization of CA125 (p = 0.03 and p = 0.044, respectively). Furthermore, we present a molecular pathway for MD/PD SC, including VEGFA, FOXM1, TPX2, BIRC5 and TOP2A, all significantly upregulated and directly interacting with TP53.

Conclusions

We have identified 21 mRNAs differentially expressed (p<0.01) between MD/PD SC, SBOT and SNO. Thirteen were differentially expressed in MD/PD SC, including ZNF385B and VEGFA correlating with survival, and FOXM1 and TPX2 with normalization of CA125. We also present a molecular pathway for MD/PD SC.     

TraK and TraB Are Conserved Outer Membrane Proteins of the Neisseria gonorrhoeae Type IV Secretion System and Are Expressed at Low Levels in Wild-Type Cells

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the medium, and this DNA is effective in transforming other gonococci via natural transformation. In addition, the T4SS is important in the initial stages of biofilm development and mediates intracellular iron uptake in the absence of TonB. To better understand the mechanism of type IV secretion in N. gonorrhoeae, we examined the expression levels and localization of two predicted T4SS outer membrane proteins, TraK and TraB, in the wild-type strain as well as in overexpression strains and in a strain lacking all of the T4SS proteins. Despite very low sequence similarity to known homologues, TraB (VirB10 homolog) and TraK (VirB9 homolog) localized similarly to related proteins in other systems. Additionally, we found that TraV (a VirB7 homolog) interacts with TraK, as in other T4SSs. However, unlike in other systems, neither TraK nor TraB required the presence of other T4SS components for proper localization. Unlike other gonococcal T4SS proteins we have investigated, protein levels of the outer membrane proteins TraK and TraB were extremely low in wild-type cells and were undetectable by Western blotting unless overexpressed or tagged with a FLAG3 triple-epitope tag. Localization of TraK-FLAG3 in otherwise wild-type cells using immunogold electron microscopy of thin sections revealed a single gold particle on some cells. These results suggest that the gonococcal T4SS may be present in single copy per cell and that small amounts of T4SS proteins TraK and TraB are sufficient for DNA secretion.     

Cyclosporin A Impairs the Secretion and Activity of ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeat)

The protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeat) cleaves multimers of von Willebrand factor, thus regulating platelet aggregation. ADAMTS13 deficiency leads to the fatal disorder thrombotic thrombocytopenic purpura (TTP). It has been observed that cyclosporin A (CsA) treatment, particularly in transplant patients, may sometimes be linked to the development of TTP. Until now, the reason for such a link was unclear. Here we provide evidence demonstrating that cyclophilin B (CypB) activity plays an important role in the secretion of active ADAMTS13. We found that CsA, an inhibitor of CypB, reduces the secretion of ADAMTS13 and leads to conformational changes in the protein resulting in diminished ADAMTS13 proteolytic activity. A direct, functional interaction between CypB (which possesses peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone functions) and ADAMTS13 is demonstrated using immunoprecipitation and siRNA knockdown of CypB. Finally, CypB knock-out mice were found to have reduced ADAMTS13 levels. Taken together, our findings indicate that cyclophilin-mediated activity is an important factor affecting secretion and activity of ADAMTS13. The large number of proline residues in ADAMTS13 is consistent with the important role of cis-trans isomerization in the proper folding of this protein. These results altogether provide a novel mechanistic explanation for CsA-induced TTP in transplant patients.     

促进原核表达蛋白可溶性的研究进展

以大肠杆菌为代表的原核表达蛋白系统具有操作简单、周期短、成本低、表达量高等优点而成为获得外源表达蛋白的首选方案.但外源蛋白在原核宿主中往往以无生物活性的包涵体形式存在,这限制了原核表达系统的广泛应用.随着对蛋白折叠动力学、参与蛋白折叠的酶和分子伴侣等认识的不断深入,科学家们通过诱导条件优化、宿主细胞改造以及使用融合标签...     

HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis

The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants.     

A Theoretical Model for the Mechanical Unfolding of Repeat Proteins

We consider the mechanical stretching of a polypeptide chain formed by multiple interacting repeats. The folding thermodynamics and the interactions among the repeats are described by the Ising model. Unfolded repeats act as soft entropic springs, whereas folded repeats respond to a force as stiffer springs. We show that the resulting force-extension curve may exhibit a pronounced force maximum corresponding to the unfolding of the first repeat. This event is followed by the unfolding of the remaining repeats, which takes place at a lower force. As the protein extension is increased, the force-extension curve of a sufficiently long repeat protein displays a plateau, where the force remains nearly constant and the protein unfolds sequentially so that the number of unfolded repeats is proportional to the extension. Such a sequential mechanical unfolding mechanism is displayed even by the repeat proteins whose thermal denaturation is highly cooperative, provided that they are long enough. By contrast, the unfolding of short repeat progressions can be cooperative.