A primer on cathepsin biology Cathepsin L transcription and translation. Substantial work has beendone to analyze the promoter regions of the human cathepsin L gene (CTSL) promoter as well as to understand the regulation of different splice variants within the 5′ untranslated region of the transcript (21,22). Of note, one of the splice variants contains a functional internal ribosomal entry site that enables ongoing translation of human cathepsin L under stress conditions, and hypoxia can shut down cap-dependent translation initiation (23). More recent work has focused on the regulation of cathepsin L alternative translation. According to the presence of different forms of cathepsin L in distinct subcellular and extracellular compartments, cathepsin L proteins can be initiated from downstream AUG sites (10), omitting the signal peptide that is normally present at the N terminus of lysosomal cathepsin L that routes the protein to the ER during its synthesis (Figure 2) (10, 24-26 Cathepsins were originally identified as proteases that act in the lysosome. Recent work has uncovered nontraditional roles for cathepsins in the extracellular space as well as in the cytosol and nucleus. There is strong evidence that subspecialized and compartmentalized cathepsins participate in many physiologic and pathophysiologic cellular processes, in which they can act as both digestive and regulatory proteases. In this review, we discuss the transcriptional and translational control of cathepsin expression, the regulation of intracellular sorting of cathepsins, and the structural basis of cathepsin activation and inhibition. In particular, we highlight the emerging roles of various cathepsin forms in disease, particularly those of the cardiac and renal systems.

Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of ϳ4 nm. Proteins near the viral membrane were arranged in overlapping lattices surrounding a disordered core. Trimeric glycoprotein spikes were in register with four underlying ribonucleoprotein densities. However, the spikes were dispensable for ribonucleoprotein lattice formation. The ribonucleoprotein particles displayed coiled shapes when released from the viral membrane. Our results contribute to the understanding of the assembly pathway used by coronaviruses and other pleomorphic viruses and provide the first detailed view of coronavirus ultrastructure.

Integrins are αβ heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin αVβ3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn2+-bound αVβ3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most αVβ3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound αVβ3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of β3, with FN9 just adjacent to the synergy site binding region of αV. We conclude that the ectodomain of αVβ3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

transformation. The type IV pili are assembled from thousands of copies of a single protein subunit, pilin. As they occur on the surfaces of virtually all Gram-negative bacteria, type IV pili and their pilin subunits represent a potentially general antibacterial target.

Integrins are a large family of heterodimeric transmembrane signaling proteins that affect diverse biological processes such as development, angiogenesis, wound healing, neoplastic transformation, and thrombosis. We report here the three-dimensional structure at 20-Å resolution of the unliganded, low-affinity state of the human platelet integrin ␣IIb␤3 derived by electron cryomicroscopy and single particle image reconstruction. The large ectodomain and small cytoplasmic domains are connected by a rod of density that we interpret as two parallel transmembrane ␣-helices. The docking of the x-ray structure of the ␣V␤3 ectodomain into the electron cryomicroscopy map of ␣IIb␤3 requires hinge movements at linker regions between domains in the crystal structure. Comparison of the putative high-and lowaffinity conformations reveals dramatic conformational changes associated with integrin activation.cell adhesion ͉ transmembrane signaling ͉ membrane proteins ͉ platelets I ntegrins are a large family of eukaryotic cell-surface receptors that mediate dynamic interactions between cells and extracellular adhesion molecules (1). They are important for the maintenance of tissue integrity and promotion of cellular migration (2) and play important roles in development, angiogenesis, wound healing, neoplastic transformation, and thrombosis (3-5). Integrins are heterodimeric type I membrane proteins composed of ␣ and ␤ subunits (6). Nineteen ␣ and eight ␤ isoforms have so far been identified in mammals (1). Each ␣ binds only a limited subset of ␤, and each ␣␤ pair manifests specific ligand-binding properties (7).␣ IIb ␤ 3 (M r 235) is one of the best characterized integrins, studied not only for its importance in thrombosis but also as a prototypical integrin (8). ␣ IIb ␤ 3 is by far the most abundant integrin on the platelet surface (40,000-80,000 copies) and is also present in internal pools (9, 10). The ␣ IIb subunit (M r 130) undergoes posttranslational proteolytic cleavage, producing a 105-kDa extracellular fragment and a 23-kDa fragment containing the single transmembrane span and a 26-residue cytoplasmic domain (8). The two chains are linked by a single disulfide bond. In contrast, ␤ 3 consists of a single polypeptide chain (M r 95), including a single transmembrane span and a 45-residue cytoplasmic domain.Previous electron microscopic (EM) images of negatively stained or metal-shadowed ␣ IIb ␤ 3 complexes revealed a globular domain Ϸ100 Å in diameter and two longer extensions of 140-170 Å (11, 12). When detergent was removed, the protein formed rosettes with an outer radius formed by the globular domain. The molecules were joined at the center by the distal ends of the extensions, suggesting that the latter structures were composed of the hydrophobic domains (11,12). Consistent with this interpretation, a soluble recombinant form of the extracellular polypeptide revealed a globular domain with shorter tails (13). A similar morphology has been seen in negative-staining studies of protein reconstituted into lipid vesicles wh...

Glycophorin A and its isolated transmembrane region (GpATM) are each known to form sequence-specific dimers in SDS micelles. Whether this behavior accurately reflects behavior in red cell membranes or lipid bilayers, however, has remained unclear. Resonance energy transfer between labeled GpATM peptides has been used to observe dimerization of GpATM in bilayers. Separate populations of GpATM peptides were labeled with 2,6-dansyl chloride as the donor chromophore and dabsyl chloride as the acceptor. Quenching of the 2,6-dansyl chloride by the dabsyl group demonstrated an association of the labeled peptides. The quenching was not affected by increases in the amount of lipid present or by unlabeled heterologous peptides but was decreased by the addition of unlabeled GpATM. GpATM was determined to form dimers by fitting the observed energy transfer for a number of donor to acceptor ratios and fitting to the expected number of donor labeled peptides in an oligomer with an acceptor as a function of oligomer number. The finding that the GpATM peptide forms helical dimers in lipid bilayers supports the idea that GpA is a dimer in the erythrocyte membrane. The resonance energy transfer approach may extend to the study of other oligomeric complexes.

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand: the 10th type III RGD-domain of wild-type fibronectin (wtFN10), or to a high affinity mutant (hFN10) that acts as a pure antagonist. Comparison of these structures revealed a central π - π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3-subunit that blocked conformational changes triggered by wtFN10, and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side-chains, or reorienting Trp1496 away from Tyr122, converted hFN10 into a partial agonist. The findings offer new insights on the mechanism of integrin activation and a basis for design of RGD-based pure antagonists.

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