A distinct concerted mechanism of structural dynamism defines activity of human serine protease HtrA3

Human HtrA3 (high-temperature requirement protease A3) is a trimeric multitasking propapoptotic serine protease associated with critical cellular functions and pathogenicity. Implicated in diseases including cancer and pre-eclampsia, its role as a tumor suppressor and potential therapeutic target cannot be ignored. Therefore, elucidating its mode of activation and regulatory switch becomes indispensable towards modulating its functions with desired effects for disease intervention. Using computational, biochemical and biophysical tools, we delineated the role of all domains, their combinations and the critical phenylalanine residues in regulating HtrA3 activity, oligomerization and specificity. Our findings underline the crucial roles of the N-terminus as well as the PDZ domain in oligomerization and formation of a catalytically competent enzyme, thus providing new insights into its structure–function coordination. Our study also reports an intricate ligand-induced allosteric switch, which redefines the existing hypothesis of HtrA3 activation besides opening up avenues for modulating protease activity favorably through suitable effector molecules.

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The crystal structure of Mycobacterium tuberculosis high-temperature requirement A protein reveals an autoregulatory mechanism

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18016217 ◽

2018 ◽

Vol 74 (12) ◽

pp. 803-809

Author(s):

Arvind Kumar Gupta ◽

Debashree Behera ◽

Balasubramanian Gopal

Keyword(s):

Crystal Structure ◽

Mycobacterium Tuberculosis ◽

High Temperature ◽

Catalytic Domain ◽

Pdz Domain ◽

Regulatory Pathways ◽

Inactive Conformation ◽

Temperature Requirement ◽

Autoregulatory Mechanism

The crystal structure of Mycobacterium tuberculosis high-temperature requirement A (HtrA) protein was determined at 1.83 Å resolution. This membrane-associated protease is essential for the survival of M. tuberculosis. The crystal structure reveals that interactions between the PDZ domain and the catalytic domain in HtrA lead to an inactive conformation. This finding is consistent with its proposed role as a regulatory protease that is conditionally activated upon appropriate environmental triggers. The structure provides a basis for directed studies to evaluate the role of this essential protein and the regulatory pathways that are influenced by this protease.

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The regulatory role of Rho GTPases and their substrates in osteoclastogenesis

Current Drug Targets ◽

10.2174/1389450121666200925150446 ◽

2020 ◽

Vol 21 ◽

Author(s):

Lin Gao ◽

Lingbo Kong ◽

Yuanting Zhao

Keyword(s):

Bone Loss ◽

Therapeutic Target ◽

Rho Gtpases ◽

Molecular Mechanisms ◽

Special Role ◽

Cellular Functions ◽

Potential Therapeutic Target ◽

Cytoskeletal Organization ◽

Osteoclastic Differentiation

: Pathological bone loss diseases (osteolysis, Paget’s diseases) are commonly caused by the over differentiation and activity of osteoclasts. The Rho GTPases family members Rac1/2 (Rac1 and Rac2) have been reported for their special role in exerting multiple cellular functions during osteoclastic differentiation, which including the most prominent function on dynamic actin cytoskeleton rearranging. Besides that, the increasing studies demonstrated the regulating effects of Rac1/2 on osteoclastic cytoskeletal organization is through the GEFs member Dock5. Although the amount of relevant studies on this topic still limited, there are several excellent studies have been reported for extensively explored the molecular mechanisms involved in Rac1/2 and Dock5 during the osteoclastogenesis regulation, as well as their role as the therapeutic target in bone loss disesases. Herein in this review, we aim to focus on recent advances studies for extensively understanding the role of Rho GTPases Rac1/2 and Dock5 in osteoclastogenesis, as well as their role as a potential therapeutic target in regulating osteoclastogenesis.

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14-3-3τ Regulates Ubiquitin-Independent Proteasomal Degradation of p21, a Novel Mechanism of p21 Downregulation in Breast Cancer

Molecular and Cellular Biology ◽

10.1128/mcb.01335-09 ◽

2010 ◽

Vol 30 (6) ◽

pp. 1508-1527 ◽

Cited By ~ 44

Author(s):

Bing Wang ◽

Kang Liu ◽

Hui-Yi Lin ◽

Naresh Bellam ◽

Shiyun Ling ◽

...

Keyword(s):

Breast Cancer ◽

Matrix Protein ◽

Proteasomal Degradation ◽

Extracellular Matrix Protein ◽

Cellular Functions ◽

Mcf7 Cells ◽

Potential Therapeutic Target ◽

Tamoxifen Response ◽

Primary Human Breast

ABSTRACT 14-3-3 proteins regulate many cellular functions, including proliferation. However, the detailed mechanisms by which they control the cell cycle remain to be fully elucidated. We report that one of the 14-3-3 isoforms, 14-3-3τ, is required for the G1/S transition through its role in ubiquitin-independent proteasomal degradation of p21. 14-3-3τ binds to p21, MDM2, and the C8 subunit of the 20S proteasome in G1 phase and facilitates proteasomal targeting of p21. This function of 14-3-3τ may be deregulated in cancer. The overexpression of 14-3-3τ is frequently found in primary human breast cancer and correlates with lower levels of p21 and shorter patient survival. Tenascin-C, an extracellular matrix protein involved in tumor initiation and progression and a known 14-3-3τ inducer, decreases p21 and abrogates adriamycin-induced G1/S arrest. It has been known that p21 is required for a proper tamoxifen response in breast cancer. We show that the overexpression of 14-3-3τ inhibits tamoxifen-induced p21 induction and growth arrest in MCF7 cells. Together, the findings of our studies strongly suggest a novel oncogenic role of 14-3-3τ by downregulating p21 in breast cancer. Therefore, 14-3-3τ may be a potential therapeutic target in breast cancer.

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Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease from Staphylococcus epidermidis

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319015055 ◽

2020 ◽

Vol 76 (1) ◽

pp. 28-40

Author(s):

Kartik Manne ◽

Sthanam V. L. Narayana

Keyword(s):

Serine Protease ◽

Staphylococcus Epidermidis ◽

Binding Pocket ◽

Glutamyl Endopeptidase ◽

Structural Components ◽

Substrate Binding Pocket ◽

N Terminus ◽

Peptide Segments ◽

Extracellular Serine Protease

Extracellular serine protease (Esp) from Staphylococcus epidermidis is a glutamyl endopeptidase that inhibits the growth and formation of S. aureus biofilms. Previously, crystal structures of the matured and active Esp have been determined. Interestingly, many of the staphylococcal glutamyl endopeptidase zymogens, including V8 from Staphylococcus aureus and Esp from S. epidermidis, contain unusually long pro-peptide segments; however, their function is not known. With the aim of elucidating the function of these pro-peptide segments, crystal structures of the Esp zymogen (Pro-Esp) and its variants were determined. It was observed that the N-terminus of the Pro-Esp crystal structure is flexible and is not associated with the main body of the enzyme, unlike in the known active Esp structure. In addition, the loops that border the putative substrate-binding pocket of Pro-Esp are flexible and disordered; the structural components that are responsible for enzyme specificity and efficiency in serine proteases are disordered in Pro-Esp. However, the N-terminal locked Pro-Esp variants exhibit a rigid substrate-binding pocket similar to the active Esp structure and regain activity. These structural studies highlight the role of the N-terminus in stabilizing the structural components responsible for the activity and specificity of staphylococcal glutamyl endopeptidases.

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Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111257118 ◽

2021 ◽

Vol 118 (35) ◽

pp. e2111257118

Author(s):

Yuki Toyama ◽

Robert W. Harkness ◽

Lewis E. Kay

Keyword(s):

High Temperature ◽

Ligand Binding ◽

Pdz Domain ◽

Nmr Studies ◽

Transverse Relaxation ◽

Temperature Requirement ◽

Biochemical Assays ◽

Substrate Concentrations ◽

Binding Substrate

The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy–based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding–incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson’s disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.

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