MK-6, a novel not-α IL-2, elicits a potent antitumor activity by improving the effector to regulatory T cell balance.

IL-2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti-immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL-2, Mutakine-6 (MK-6), with reduced IL-2Rα-binding capability. MK-6 induced comparable cell growth potential toward IL-2Rßγ-positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL-2, in vivo administration of MK-6 produced minimal adverse effects. Using CT26 and B16F10-syngeneic tumor models, we found MK-6 was highly efficacious on tumor regression. Serum albumin conjugation to MK-6 prolonged in vivo half-life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor-infiltrating leukocytes analysis revealed that albumin-fused MK-6 increased the ratio of effector CD8+ T cells to CD4+ Treg cells. These results demonstrated that MK-6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation.

Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction.

Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1ΔCT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC-RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1ΔCT-expressed muscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.

Angiotensin II activates CaV 1.2 Ca2+ channels through ß-arrestin2 and casein kinase 2 in mouse immature cardiomyocytes.

KEY POINTS: Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians. Here we show that AngII increases twitch Ca2+ transients of mouse immature but not mature cardiomyocytes by robustly activating CaV 1.2 L-type Ca2+ channels through a novel signalling pathway involving angiotensin type 1 (AT1 ) receptors, ß-arrestin2 and casein kinase 2. A ß-arrestin-biased AT1 receptor agonist, TRV027, was as effective as AngII in activating L-type Ca2+ channels. Our results help understand the molecular mechanism by which AngII regulates the perinatal circulation and also suggest that ß-arrestin-biased AT1 receptor agonists may be valuable therapeutics for paediatric heart failure. ABSTRACT: Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system, plays important roles in cardiovascular regulation in the perinatal period. Despite the well-known stimulatory effect of AngII on vascular contraction, little is known about regulation of contraction of the immature heart by AngII. Here we found that AngII significantly increased the peak amplitude of twitch Ca2+ transients by robustly activating L-type CaV 1.2 Ca2+ (CaV 1.2) channels in mouse immature but not mature cardiomyocytes. This response to AngII was mediated by AT1 receptors and ß-arrestin2. A ß-arrestin-biased AT1 receptor agonist was as effective as AngII in activating CaV 1.2 channels. Src-family tyrosine kinases (SFKs) and casein kinase 2α'ß (CK2α'ß) were sequentially activated when AngII activated CaV 1.2 channels. A cyclin-dependent kinase inhibitor, p27Kip1 (p27), inhibited CK2α'ß, and AngII removed this inhibitory effect through phosphorylating tyrosine 88 of p27 via SFKs in cardiomyocytes. In a human embryonic kidney cell line, tsA201 cells, overexpression of CK2α'ß but not c-Src directly activated recombinant CaV 1.2 channels composed of C-terminally truncated α1C , the distal C-terminus of α1C , ß2C and α2 δ1 subunits, by phosphorylating threonine 1704 located at the interface between the proximal and the distal C-terminus of CaV 1.2α1C subunits. Co-immunoprecipitation revealed that CaV 1.2 channels, CK2α'ß and p27 formed a macromolecular complex. Therefore, stimulation of AT1 receptors by AngII activates CaV 1.2 channels through ß-arrestin2 and CK2α'ß, thereby probably exerting a positive inotropic effect in the immature heart. Our results also indicated that ß-arrestin-biased AT1 receptor agonists may be used as valuable therapeutics for paediatric heart failure in the future.

ESCRT-0 protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is targeted to endosomes independently of signal-transducing adaptor molecule (STAM) and the complex formation with STAM promotes its endosomal dissociation.

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.

Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal.

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor ß (IL-2Rß) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rß and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rß and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rß together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rß. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor ß (IL-2Rß). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor α (IL-4Rα), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2Rß. Examinations of the Hrs binding regions of the receptors reveal that a hydrophobic amino acid cluster in both IL-2Rß and IL-4Rα is essential for the binding. Whereas the wild-type receptors are delivered to LAMP1-positive late endosomes, mutant receptors lacking the hydrophobic amino acid cluster are sorted to lysobisphosphatidic acid-positive late endosomes rather than LAMP1-positive late endosomes. We also show that the degradation of these mutant receptors is attenuated. Accordingly, Hrs functions during ubiquitin-independent endosomal sorting of the receptors by recognizing the hydrophobic amino acid cluster. These findings suggest the existence of a group of cargo proteins that have this hydrophobic amino acid cluster as a ubiquitin-independent sorting signal.

EF-1α and mitochondrial ATP synthase ß chain: alteration of their expression in encystment-induced Colpoda cucullus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus.

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 x 10(4) cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.

A low-frequency IL4R locus variant in Japanese patients with intravenous immunoglobulin therapy-unresponsive Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis which may be associated with coronary artery aneurysms. A notable risk factor for the development of coronary artery aneurysms is resistance to intravenous immunoglobulin (IVIG) therapy, which comprises standard treatment for the acute phase of KD. The cause of IVIG resistance in KD is largely unknown; however, the contribution of genetic factors, especially variants in immune-related genes, has been suspected. METHODS: To explore genetic variants related to IVIG-unresponsiveness, we designated KD patients who did not respond to both first and second courses of IVIG therapy as IVIG-unresponsive patients. Using genomic DNA from 30 IVIG-unresponsive KD patients, we performed pooled genome sequencing targeting 39 immune-related cytokine receptor genes. RESULTS: The single nucleotide variant (SNV), rs563535954 (located in the IL4R locus), was concentrated in IVIG-unresponsive KD patients. Individual genotyping showed that the minor allele of rs563535954 was present in 4/33 patients with IVIG-unresponsive KD, compared with 20/1063 individuals in the Japanese genome variation database (odds ratio = 7.19, 95% confidence interval 2.43-21.47). Furthermore, the minor allele of rs563535954 was absent in 42 KD patients who responded to IVIG treatment (P = 0.0337), indicating that a low-frequency variant, rs563535954, is associated with IVIG-unresponsiveness in KD patients. Although rs563535954 is located in the 3'-untranslated region of IL4R, there was no alternation in IL4R expression associated with the mior allele of rs563535954. However, IVIG-unresponsive patients that exhibited the minor allele of rs563535954 tended to be classified into the low-risk group (based on previously reported risk scores) for prediction of IVIG-resistance. Therefore, IVIG-unresponsiveness associated with the minor allele of rs563535954 might differ from IVIG-unresponsiveness associated with previous risk factors used to evaluate IVIG-unresponsiveness in KD. CONCLUSION: These findings suggest that the SNV rs563535954 could serve as a predictive indicator of IVIG-unresponsiveness, thereby improving the sensitivity of risk scoring systems, and may aid in prevention of coronary artery lesions in KD patients.

AMSH, an ESCRT-III associated enzyme, deubiquitinates cargo on MVB/late endosomes.

The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport. Here, we purified an AMSH-binding protein, CHMP3, which is an ESCRT-III subunit. ESCRT-III functions on maturing endosomes, indicating AMSH might also play a role in MVB/late endosomes. Expression of an AMSH mutant lacking CHMP3-binding ability resulted in aberrant endosomes with accumulations of ubiquitinated cargo. Nevertheless, CHMP3-binding capability was not essential for AMSH's in vitro DUB activity or its endosomal localization, suggesting that, in vivo, the deubiquitination of endosomal cargo is CHMP3-dependent. Ubiquitinated cargo also accumulated on endosomes when catalytically inactive AMSH was expressed or AMSH was depleted. These results suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes.

Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer.

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.

Morphogenetic and molecular analyses of cyst wall components in the ciliated protozoan Colpoda cucullus Nag-1.

The cyst wall of the resting cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these proteins revealed that the 45-kDa protein (p45) was elongation factor Tu (EF-Tu). Immunofluorescence microscopy with an anti-EF-Tu polyclonal antibody showed that Colpoda EF-Tu (p45) was localized in the lepidosomes. The lepidosomes were stained vividly with Congo red, which is bound to the stacked ß-sheets of amyloid protofibrils. In the presence of puromycin, no cyst wall components including lepidosomes were formed, indicating that cyst wall formation requires synthesis of proteins including EF-Tu. Electron microscopy of encysting cells implied that vesicles which were presumably budded from endoplasmic reticula possibly fuse with a lepidosome-precursor vacuole containing electron-dense fine particles or fibrous structures, and followed by the subsequent fusion with other electron-lucent granules.

Synthesis and structure-activity relationships of novel parenteral carbapenems, CS-023 (R-115685) and related compounds containing an amidine moiety.

In order to design a new parenteral 1beta-methylcarbapenem antibiotic which has a broad antibacterial spectrum and improved plasma half-life, a series of 1beta-methylcarbapenems with 5-substituted pyrrolidine-3-ylthio groups including an amidine moiety at the C-2 position have been synthesized and structure-activity relationships were investigated. Among those carbapenem derivatives, CS-023 (R-115685) showed a broad spectrum and excellent antibacterial activity against Gram-positive and Gram-negative bacteria. This compound also showed sufficient dehydropeptidase-I (DHP-I) stability and high urinary recovery in animals after subcutaneous administration without cilastatin, a DHP-I inhibitor. Based on these characteristics, CS-023 was selected for further study.

Excystment-dependent alteration of protein expression in terrestrial ciliate Colpoda cucullus.

Protein expression during the excystment of Colpoda cucullus was studied by SDS-PAGE. The expression levels of 60-, 50- and 49-kDa proteins were markedly changed from the early to later stage of excystment. The 60-kDa protein (p60) was temporarily expressed first, and its expression was inhibited by actinomycin D. LC-MS/MS analysis showed that the amino acid sequences of p60 partially coincided with those of the Paramecium tetraurelia unnamed protein homologous to DEAD-box RNA helicase. These results suggest that p60 expression is enhanced by transcriptional regulation and may be involved in initiating the molecular events leading to cellular morphogenesis.

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins.

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them. The p33 obtained from isolated macronuclei was tentatively identified as ribosomal P0 protein by LC-MS/MS analysis. In addition, among the encystment-specific phosphoproteins obtained by phosphate-affinity chromatography, the 29-, 31-, and 33-kDa proteins (p29, p31, and p33) were tentatively identified as ribosomal P0 protein, whereas the 24-kDa phosphoprotein (p24) was tentatively identified as ribosomal S5 protein.

Ubiquitin-independent binding of Hrs mediates endosomal sorting of the interleukin-2 receptor beta-chain.

Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor beta (IL-2Rbeta). We found a direct interaction between bacterially expressed IL-2Rbeta and Hrs in GST pull-down assays, indicating that their binding is independent of ubiquitin. Trafficking and degradation assays revealed that, similarly to wild-type IL-2Rbeta, an IL-2Rbeta mutant lacking all the cytoplasmic lysine residues is sorted from Hrs-positive early endosomes to LAMP1-positive late endosomes, resulting in degradation of the receptor. By contrast, an IL-2Rbeta mutant lacking the Hrs-binding region passes through early endosomes and is mis-sorted to compartments positive for the transferrin receptor. The latter mutant exhibits attenuated degradation. Taken together, these results indicate that precise sorting of IL-2Rbeta from early to late endosomes is mediated by Hrs, a known sorting component of the ubiquitin-dependent machinery, in a manner that is independent of UIM-ubiquitin binding.

Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line.

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites (+/-5kb), near CpG islands (+/-1kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.

Determination of the cleavage site of the presequence by mitochondrial processing peptidase on the substrate binding scaffold and the multiple subsites inside a molecular cavity.

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the -2 position (P(2)). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, alpha-MPP and beta-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat beta-MPP interacting with the P(2) arginine cause a shift in the processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P(2) arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P(2) arginine but also the potential C-terminal cleavage site without a P(2) arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of alpha-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P(2) arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from beta- to alpha-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P(2) arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.