HtrA1 activation is driven by an allosteric mechanism of inter-monomer communication

Cortés-Cabrera, Álvaro; Roth, Doris; Topp, Andreas; Delobel, Frédéric; Stucki, Corinne; Chen, Chia-yi; Jakob, Peter; Bánfai, Balázs; Dunkley, Tom; Schilling, Oliver; Huber, Sylwia; Iacone, Roberto; Petrone, Paula

doi:10.1038/s41598-017-14208-z

Cited by 24 publications

(24 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In line with these observations, deletion of the trimerization motif in human HtrA2 showed that the formation of the homotrimer is essential for precise function of the enzyme including its proteolytic activity ( Li et al, 2002 ; Nam et al, 2006 ). Recently, it was experimentally also shown that trimerization plays a fundamental role for the activation of human HtrA1 by an allosteric mechanism ( Cabrera et al, 2017 ). Further, computational studies of DegS suggest that disassembly of a DegS trimer inhibits proteolytic activity ( Lu et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

et al. 2018

View full text Add to dashboard Cite

The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the virulence of the gastric pathogen Helicobacter pylori. Secreted H. pylori HtrA (HtrAHp) cleaves various junctional proteins such as E-cadherin disrupting the epithelial barrier, which is crucial for bacterial transmigration across the polarized epithelium. Recent studies indicated the presence of two characteristic HtrAHp forms of 55 and 52 kDa (termed p55 and p52, respectively), in worldwide strains. In addition, p55 and p52 were produced by recombinant HtrAHp, indicating auto-cleavage. However, the cleavage sites and their functional importance are yet unclear. Here, we determined the amino-terminal ends of p55 and p52 by Edman sequencing. Two proteolytic cleavage sites were identified (H46/D47 and K50/D51). Remarkably, the cleavage site sequences are conserved in HtrAHp from worldwide isolates, but not in other Gram-negative pathogens, suggesting a highly specific assignment in H. pylori. We analyzed the role of the amino-terminal cleavage sites on activity, secretion and function of HtrAHp. Three-dimensional modeling suggested a trimeric structure and a role of amino-terminal processing in oligomerization and regulation of proteolytic activity of HtrAHp. Furthermore, point and deletion mutants of these processing sites were generated in the recently reported Campylobacter jejuni ΔhtrA/htrAHp genetic complementation system and the minimal sequence requirements for processing were determined. Polarized Caco-2 epithelial cells were infected with these strains and analyzed by immunofluorescence microscopy. The results indicated that HtrAHp processing strongly affected the ability of the protease to disrupt the E-cadherin-based cell-to-cell junctions. Casein zymography confirmed that the amino-terminal region is required for maintaining the proteolytic activity of HtrAHp. Furthermore, we demonstrated that this cleavage influences the secretion of HtrAHp in the extracellular space as an important prerequisite for its virulence activity. Taken together, our data demonstrate that amino-terminal cleavage of HtrAHp is conserved in this pathogen and affects oligomerization and thus, secretion and regulatory activities, suggesting an important role in the pathogenesis of H. pylori.

show abstract

Section: Discussionmentioning

confidence: 99%

Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Among the other missense HTRA1s observed in the symptomatic carriers, four of the five missense HTRA1s had a mutation in the LD domain. The simulation analysis revealed that these mutations induced an instability in the helical structure of the LD loop (16). For the L3 domain, R302 is the only portion observed in the symptomatic carrier.…”

Section: Discussionmentioning

confidence: 99%

“…For the L3 domain, R302 is the only portion observed in the symptomatic carrier. R302 is essential for inter-monomer communication and substrate binding (16). Thus, a mutation in the LD or L3 domain that interferes with signal transduction between the monomers result in inhibition of the WT activity.…”

Section: Discussionmentioning

confidence: 99%

HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade

et al. 2019

View full text Add to dashboard Cite

Background: Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Most carriers for HTRA1 mutations are asymptomatic, but more than 10 mutations have been reported in symptomatic carriers. The molecular differences between the mutations identified in symptomatic carriers and mutations identified only in CARASIL patients are unclear. HTRA1 is a serine protease that forms homotrimers, with each HTRA1 subunit activating the adjacent HTRA1 via the sensor domain of loop 3 (L3) and the activation domain of loop D (LD). Previously, we analyzed four HTRA1 mutant proteins identified in symptomatic carriers and found that they were unable to form trimers or had mutations in the LD or L3 domain. The mutant HTRA1s with these properties are presumed to inhibit trimer-dependent activation cascade. Indeed, these mutant HTRA1s inhibited wild-type (WT) protease activity. In this study, we further analyzed 15 missense HTRA1s to clarify the molecular character of mutant HTRA1s identified in symptomatic carriers. Methods: We analyzed 12 missense HTRA1s identified in symptomatic carriers (hetero-HTRA1) and three missense HTRA1s found only in CARASIL (CARASIL-HTRA1). The protease activity of the purified recombinant mutant HTRA1s was measured using fluorescein isothiocyanate-labeled casein as substrate. Oligomeric structure was evaluated by size-exclusion chromatography. The protease activities of mixtures of WT with each mutant HTRA1 were also measured. Results: Five hetero-HTRA1s had normal protease activity and were excluded from further analysis. Four of the seven hetero-HTRA1s and one of the three CARASIL-HTRA1s were unable to form trimers. The other three hetero-HTRA1s had mutations in the LD domain. Together with our previous work, 10 of 11 hetero-HTRA1s and two of six CARASIL-HTRA1s were either defective in trimerization or had mutations in the LD or L3 domain ( P = 0.006). By contrast, eight of 11 hetero-HTRA1s and two of six CARASIL-HTRA1 inhibited WT protease activity ( P = 0.162). Conclusions: HTRA1 mutations identified in symptomatic carriers have the property of interfering the trimer-dependent activation cascade of HTRA1.

show abstract

“…Recently, two different mechanisms were proposed to underlie the dominant-negative effects of HTRA1 mutations (4,11): the inhibition of trimer formation, and the inhibition of the wild-type HTRA1 activity, observed in several missense mutations located in the sensor domain of loop 3 (L3) or the activation domain of loop D (LD) of HTRA1. These two domains play an essential role in the normal protease activity of HTRA1 (10,12). Among the previously reported missense mutations, p.R302 Q, which replaces the evolutionarily well-conserved arginine with glutamine, is the only mutation located in L3.…”

Section: Discussionmentioning

confidence: 99%

Cerebral Small Vessel Disease Related to a Heterozygous Nonsense Mutation in <i>HTRA1</i>

et al. 2020

View full text Add to dashboard Cite

Homozygous or compound heterozygous mutations in the high-temperature requirement A serine protease 1 gene (HTRA1) cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a very rare hereditary cerebral small-vessel disease (SVD). Recently, the relationship between some heterozygous HTRA1 mutations, most of which are missense, and the occurrence of cerebral SVD has been reported. We herein report a patient with cerebral SVD carrying a heterozygous nonsense p.R302X mutation in HTRA1. This patient had a family history of cerebral infarction. This report suggests that a heterozygous p.R302X mutation in HTRA1 causes an autosomal dominant cerebral SVD.

show abstract

HtrA1 activation is driven by an allosteric mechanism of inter-monomer communication

Cited by 24 publications

References 38 publications

Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

HTRA1 Mutations Identified in Symptomatic Carriers Have the Property of Interfering the Trimer-Dependent Activation Cascade

Cerebral Small Vessel Disease Related to a Heterozygous Nonsense Mutation in <i>HTRA1</i>

Contact Info

Product

Resources

About