Inhibitor SourceBook™ Second Edition

Proteases
Calpain Inhibitors
Calpains belong to a family of calcium-dependent thiolproteases
that proteolyze a wide variety of cytoskeletal,
membrane-associated, and regulatory proteins. Fifteen
gene products of the calpain family are reported in
mammals, which are classified as nine typical and six
atypical calpains. Typical calpains are characterized
by a C-terminal Ca 2+ -binding domain that includes EFhand
motifs, while atypical calpains lack this region,
but often contain additional domains. Calpains do not
generally function as destructive proteases, but act as
calcium-dependent modulators that remove limited
portions of protein substrates. Calpains respond to
Ca 2+ signals by cleaving specific proteins, frequently
components of signaling cascades, thereby irreversibly
modifying their function. Two major isoforms of calpain
are reported in mammals, calpain-1 (m-form) and
calpain-2 (m-form). They are constitutively expressed
in all tissues and differ in their calcium requirement
for activation (~50 mM for calpain-1 and ~500 mM for
calpain-2) and contain several calcium-binding sites,
which allosterically affect the enzyme activity.
Calpain-1 and -2 exhibit about 55-65% sequence
homology and are composed of an 80 kDa and a 30 kDa
subunit. The 80 kDa subunit has the catalytic site and
is unique to each isozyme, whereas the 30 kDa unit is
the regulatory subunit and is common to both calpain-
1 and -2. The 80 kDa subunit consists of four domains
(I-IV) and the 30 kDa unit has 2 domains (V and VI).
Domain I is partially removed during autolysis. Domain
II is the protease domain, which is structurally similar
to the catalytic domain of other cysteine proteases. It
contains a Cys-His-Asn triad characteristic of cysteine
proteases. Domain III exhibits a homology with typical
calmodulin binding proteins and interacts with calcium
binding domains (IV and VI) and frees domain II for
protease activity. Domain IV is a Ca 2+ -binding domain
structurally similar to calmodulin. Domain V contains
a hydrophobic region and is essential for calpain
interaction with membranes. Domain VI has five EFhands.
There are two EF-hand pairs, one pair (EF1-EF2)
displays an ‘open’ conformation and the other (EF3-EF4)
a ‘closed’ conformation. The fifth EF-hand (EF5) is in a
‘closed’ conformation. Both, calpain-1 and -2 associate
non-covalently with domain V and domain VI. Several
putative substrates of calpain-1 and -2 are known
that are cleaved by both isoforms, but with different
efficiencies. Recently, determinants for calpain-1 and
-2 have been analyzed and it is shown that amino acid
preferences extend over 11 residues around the scissile
bond. Calpains prefer Leu, Thr, Val in the P2 position,
Lys, Tyr, Arg in the P1 position, and proline in the region
flanking the P2 - P’1 segment.
Calpain-3, another typical calpain was first described
as a skeletal muscle-specific calpain isoform. However,
subsequent studies have shown its presence in several
other tissues. It is a 94 kDa enzyme that contains
821 amino acids. Structurally calpain-3 is similar
to calpain-1 and -2, however, it has an additional Nterminal
sequence of 20-30 amino acids (NS). Also, its
domain I exhibits less than 20% homology to calpain-1
and -2. Calpain-3 includes two characteristic amino
acid stretches: one between the catalytic cysteine
and histidine (IS1) and the other (IS2) upstream of the
first EF hand motif of calcium-binding domain IV.
A characteristic feature of calpain 3 is that it is not
inhibited by calpastatin. While conventional calpains
localize mainly in the cytosol or at the inner face of
the plasma membrane, calpain-3 is detected within the
nuclei of skeletal muscle cells and in the N2 region of
myofibrils.
More recently, attention has been focused on the
pathological significance of calcium accumulation in
the central nervous system following cerebral ischemia
and traumatic brain injury. Overactivation of NMDA,
kainate, and AMPA receptors in the brain leads to
sustained influx of Ca 2+ through the voltage-gated
calcium channels. Disturbances in calcium homeostasis
result in the activation of several calcium-dependent
enzymes including calpains. Overexpression of calpains
has been positively linked to both acute and chronic
neurodegenerative processes including ischemia,
trauma, and Alzheimer’s disease. In Alzheimer’s disease
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