Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates.

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.

Chemical biology approaches reveal conserved features of a C-terminal processing PDZ protease.

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C-terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram-negative bacteria. While several HtrA proteases have been extensively characterized, many features of C-terminal processing proteases such as tail-specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid-based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed.

Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3.

Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for subtilases in plant biology. We report the first structure of a plant subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal seven-stranded jelly-roll fibronectin (Fn) III-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although Ca(2+)-binding sites are conserved and critical for stability in other subtilases, SBT3 was found to be Ca(2+)-free and its thermo stability is Ca(2+)-independent.

Selectivity profiling of DegP substrates and inhibitors.

Protein quality control factors are involved in many key physiological processes and severe human diseases that are based on misfolding or amyloid formation. Prokaryotic representatives are often virulence factors of pathogenic bacteria. Therefore, protein quality control factors represent a novel class of drug targets. The bacterial serine protease DegP, belonging to the widely conserved family of HtrA proteases, exhibits unusual structural and functional plasticity that could be exploited by small molecule modulators. However, only one weak synthetic peptide substrate and no inhibitors are available to date. We report the identification of a potent heptameric pNA-substrate and chloromethyl ketone based inhibitors of DegP. In addition, specificity profiling resulted in the identification of one strong inhibitor and a potent substrate for subtilisin as well as a number of specific elastase substrates and inhibitors.

Allosteric regulation of proteases.

Allostery is a basic principle of control of enzymatic activities based on the interaction of a protein or small molecule at a site distinct from an enzyme's active center. Allosteric modulators represent an alternative approach to the design and synthesis of small-molecule activators or inhibitors of proteases and are therefore of wide interest for medicinal chemistry. The structural bases of some proteinaceous and small-molecule allosteric protease regulators have already been elucidated, indicating a general mechanism that might be exploitable for future rational design of small-molecule effectors.

E. coli LoiP (YggG), a metalloprotease hydrolyzing Phe-Phe bonds.

YggG is a conserved lipoprotein localized to the outer membrane of Gram negative bacteria. Even though the expressed open reading frame has been identified previously, the Escherichia coli protein remained uncharacterized. We report that YggG of E. coli is a metalloprotease that cleaves its targets preferentially between Phe-Phe residues. Since the yggG promoter is upregulated when bacteria are subjected to media of low osmolarity, YggG was named LoiP (low osmolarity induced protease). LoiP has an intramolecular disulfide (S-S) bond that is formed even in the absence of the periplasmic oxido-reductase DsbA and proper membrane localization of LoiP can depend on another putative metalloprotease, YfgC.

Conversion of a regulatory into a degradative protease.

The PDZ protease DegS senses mislocalized outer membrane proteins and initiates the sigmaE pathway in the bacterial periplasm. This unfolded protein response pathway is activated by processing of the anti-sigma factor RseA by DegS and other proteases acting downstream of DegS. DegS mediates the rate-limiting step of sigma E induction and its activity must be highly specific and tightly regulated. While DegS is structurally and biochemically well studied, the determinants of its pronounced substrate specificity are unknown. We therefore performed swapping experiments by introducing elements of the homologous but unspecific PDZ protease DegP. Introduction of loop L2 of DegP into DegS converted the enzyme into a non-specific protease, while swapping of PDZ domains did not. Therefore, loop L2 of the protease domain is a key determinant of substrate specificity. Interestingly, swapping of loop L2 did not affect the tight regulation of DegS. In addition, the combined introduction of loop L2 and PDZ domain 1 of DegP into DegS converted DegS even further into a DegP-like protease. These and other data suggest that homologous enzymes with distinct activities and regulatory features can be converted by simple genetic modifications.

Determinants of structural and functional plasticity of a widely conserved protease chaperone complex.

Channeling of misfolded proteins into repair, assembly or degradation pathways is often mediated by complex and multifunctional cellular factors. Despite detailed structural information, the underlying regulatory mechanisms governing these factors are not well understood. The extracytoplasmic heat-shock factor DegP (HtrA) is a well-suited model for addressing mechanistic issues, as it is regulated by the common mechanisms of allostery and activation by oligomerization. Site-directed mutagenesis combined with refolding and oligomerization studies of chemically denatured DegP revealed how substrates trigger the conversion of the resting conformation into the active conformation. Binding of specific peptides to PDZ domain-1 causes a local rearrangement that is allosterically transmitted to the substrate-binding pocket of the protease domain. This activated state readily assembles into larger oligomeric particles, thus stabilizing the catalytically active form and providing a degradation cavity for protein substrates. The implications of these data for the mechanism of protein quality control are discussed.

Peptidic small molecule activators of the stress sensor DegS.

Bacterial DegS is a regulatory protease that acts as a molecular stress sensor and initiates a periplasmic stress response pathway. Upon binding of misfolded proteins to its PDZ domain, the protease domain of DegS is allosterically activated, thereby initiating a signal cascade that results in the elevated expression of protein quality control factors. Although the structural basis of this activation mode has been elucidated previously, it is not yet fully understood if binding to the PDZ domain is sufficient for protease domain activation or if secondary interactions with the protease domain are required. Here, we demonstrate that tripeptidic small molecule activators which only bind to the PDZ domain are sufficient to trigger DegS activation. Furthermore, we show that the hydrophobicity of the peptidic small molecule activators is a critical determinant for efficient activation.

Structure, function and regulation of the conserved serine proteases DegP and DegS of Escherichia coli.

Two members of the widely conserved HtrA family of serine proteases, DegP and DegS, are key players in extracytoplasmic protein quality control. The underlying mechanisms of their main functions in stress sensing, regulation and protection during the unfolded protein response are discussed.