Efficient expression and purification of recombinant human μ‐calpain using an <i><scp>E</scp>scherichia coli</i> expression system

Hata, Shoji; Kitamura, Fujiko; Sorimachi, Hiroyuki

doi:10.1111/gtc.12071

Cited by 7 publications

(3 citation statements)

References 43 publications

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“…Cysteine 115 is in the catalytic site of calpain-1, and its mutation in recombinant human calpain-1 completely eliminated calpain-1 activity (Hata et al, 2013), indicating that the C115Y mutation in dog is a calpain-1 null mutation. In humans, we identified three consanguineous families that were homozygous in affected individuals around the CAPN1 region and exome sequencing identified mutations in the CAPN1 gene, and one recessive family with CAPN1 compound heterozygous mutations.…”

Section: Discussionmentioning

confidence: 99%

Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

et al. 2016

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SUMMARY A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous CAPN1 null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knock-out (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1 mediated cleavage of PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis, and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans.

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Section: Discussionmentioning

confidence: 99%

Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

et al. 2016

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“…So what role could SERA5 play? We actually believe it unsurprising that wild-type SERA5 does not exhibit detectable peptidase activity, since experimental replacement with Ser of the nucleophilic Cys in papain, cathepsin L, cathepsin S and the related clan CA enzyme μ-calpain abolishes enzyme activity (Clark and Lowe, 1978 ; Coulombe et al ., 1996 ; Turkenburg et al ., 2002 ; Hata et al ., 2013 ), and numerous examples of active-site Cys-to-Ser mutations of other cysteine proteases including the clan C1B protease bleomycin hydrolase (O’Farrell et al ., 1999 ) and several clan CD caspases (e.g. Van Criekinge et al ., 1996 ) have shown that this mutation invariably results in loss of catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

Plasmodium falciparum SERA5 plays a non‐enzymatic role in the malarial asexual blood‐stage lifecycle

Stallmach¹,

Kavishwar²,

Withers‐Martinez³

et al. 2015

Molecular Microbiology

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The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparum PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.

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“…Human cardiac TnT (Merck, 648484-100 μg, purity 95%), recombinant human TnT type 2 (Novus Bio, NBC-1-28765-0.1mg), porcine calpain-1 (porcine erythrocyte, 208712-1mg, Merck Millipore) and domain 1 fragment of CAST (CAST-d1) (TaKaRa Bio) were purchased as purified proteins. Recombinant human calpain-2 and proteolytically inactive mutant calpains, calpain-1:CS (C1:CS) and calpain-2:CS (C2:CS), were produced and purified as described previously [ 20 , 21 ]. Specific activity of calpains was defined by casein assay (100–350 U/mg) [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

Calpain-2 participates in the process of calpain-1 inactivation

et al. 2020

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Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca2+-dependent manner. However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2–specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulator of calpain-1, a mechanism that may be applicable to other calpain species as well.

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Efficient expression and purification of recombinant human μ‐calpain using an Escherichia coli expression system

Cited by 7 publications

References 43 publications

Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

Plasmodium falciparum SERA5 plays a non‐enzymatic role in the malarial asexual blood‐stage lifecycle

Calpain-2 participates in the process of calpain-1 inactivation

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