Differential effects of ‘resurrecting' Csp pseudoproteases during Clostridioides difficile spore germination

AbstractClostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the vertebrate gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently determined crystal structure of CspC revealed that its degenerate site residues align closely with the catalytic triad of CspB, so in this study we tested whether the ancestral protease activity of the CspC and CspA pseudoproteases could be “resurrected.” Restoring the catalytic triad to these pseudoproteases failed to resurrect their protease activity, although the mutations differentially affected the stability and function of these pseudoproteases. Degenerate site mutations destabilized CspC and impaired spore germination without impacting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since close homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results imply that bioinformatics predictions of enzyme activity may overlook pseudoenzymes in some cases.

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The interplay of the type II TA system with other processes

10.7287/peerj.preprints.27770v2 ◽

2019 ◽

Author(s):

Wei Wen-ping ◽

Jia Wan Zhong ◽

Yang Min

Keyword(s):

Mycobacterium Tuberculosis ◽

Environmental Stress ◽

Regulatory Mechanism ◽

Phage Infection ◽

Type Ii ◽

System Research ◽

Cellular Processes ◽

The Stability ◽

And Function ◽

Insight Into

The type II toxin antitoxin (TA) system is the most well-studied TA system and is widely distributed in bacteria, especially pathogens such as Mycobacterium tuberculosis. Type II TA system plays an important role in many cellular processes, including maintaining the stability of mobile genetic elements, and bacterial altruistic suicide in response to nutritional starvation, environmental stress and phage infection. Interactions between toxin proteins and antitoxin proteins are critical for the regulation and function of type II TA systems; indeed, the understanding of their function is mainly derived from interaction and regulation of paired TA system proteins. Nonetheless, investigating interaction between unpaired TA system proteins, and the interaction between TA system proteins and other functional proteins, are becoming more common and have provided new insight into the complexity of its regulatory mechanism. In this review, we outlined the cross-interaction between TA system proteins, and the interaction between TA system proteins and other functional proteins, and we are trying to explain novel mechanism of TA system in the regulation of cellular activities. On this basis, we further discussed the knowledge and physiological implications of the relevant aspects of TA system research.

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Regulatory effects of post-translational modifications on zDHHC S-acyltransferases

Journal of Biological Chemistry ◽

10.1074/jbc.rev120.014717 ◽

2020 ◽

Vol 295 (43) ◽

pp. 14640-14652

Author(s):

Filip Zmuda ◽

Luke H. Chamberlain

Keyword(s):

Drug Targets ◽

Acyl Chain ◽

Fatty Acyl ◽

Fatty Acyl Chain ◽

Physiological Importance ◽

Cellular Processes ◽

Cell Growth And Differentiation ◽

Regulatory Effects ◽

The Stability ◽

And Function

The human zDHHC S-acyltransferase family comprises 23 enzymes that mediate the S-acylation of a multitude of cellular proteins, including channels, receptors, transporters, signaling molecules, scaffolds, and chaperones. This reversible post-transitional modification (PTM) involves the attachment of a fatty acyl chain, usually derived from palmitoyl-CoA, to specific cysteine residues on target proteins, which affects their stability, localization, and function. These outcomes are essential to control many processes, including synaptic transmission and plasticity, cell growth and differentiation, and infectivity of viruses and other pathogens. Given the physiological importance of S-acylation, it is unsurprising that perturbations in this process, including mutations in ZDHHC genes, have been linked to different neurological pathologies and cancers, and there is growing interest in zDHHC enzymes as novel drug targets. Although zDHHC enzymes control a diverse array of cellular processes and are associated with major disorders, our understanding of these enzymes is surprisingly incomplete, particularly with regard to the regulatory mechanisms controlling these enzymes. However, there is growing evidence highlighting the role of different PTMs in this process. In this review, we discuss how PTMs, including phosphorylation, S-acylation, and ubiquitination, affect the stability, localization, and function of zDHHC enzymes and speculate on possible effects of PTMs that have emerged from larger screening studies. Developing a better understanding of the regulatory effects of PTMs on zDHHC enzymes will provide new insight into the intracellular dynamics of S-acylation and may also highlight novel approaches to modulate S-acylation for clinical gain.

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Legionella pneumophila regulates host cell motility by targeting Phldb2 with a 14-3-3ζ-dependent protease effector

10.1101/2021.09.06.459093 ◽

2021 ◽

Author(s):

Lei Song ◽

Jingjing Luo ◽

Dan Huang ◽

Yunhao Tan ◽

Yao Liu ◽

...

Keyword(s):

Cell Migration ◽

Cell Motility ◽

Host Cell ◽

Legionella Pneumophila ◽

Protease Activity ◽

Regulatory Protein ◽

Bacterial Pathogen ◽

Ph Domain ◽

Cellular Processes ◽

Common Target

The cytoskeleton network of eukaryotic cells is essential for diverse cellular processes, including vesicle trafficking, cell motility and immunity, thus is a common target for bacterial virulence factors. A number of effectors from the bacterial pathogen Legionella pneumophila have been shown to modulate the function of host actin cytoskeleton to construct the Legionella-containing vacuole (LCV) permissive for its intracellular replication. In this study, we identified the Dot/Icm effector Lem8 (Lpg1290) as a protease that interferes with host motility. We show that the protease activity of Lem8 is catalyzed by a Cys-His-Asp motif known to be associated with diverse biochemical activities. Intriguingly, we found that Lem8 interacts with the host regulatory protein 14-3-3ζ, which activates its protease activity. Furthermore, Lem8 undergoes self-cleavage in a process that requires 14-3-3ζ. We identified the PH domain-containing protein Phldb2 involved in cell migration as a target of Lem8 and demonstrate that Lem8 plays a role in the inhibition of host cell migration. Our results reveal a novel mechanism of inhibiting host cell motility by L. pneumophila for its virulence.

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Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence

Biochemical Journal ◽

10.1042/bj20101865 ◽

2011 ◽

Vol 436 (2) ◽

pp. 291-303 ◽

Cited By ~ 7

Author(s):

Domina Falcone ◽

Matthew P. Henderson ◽

Hendrik Nieuwland ◽

Christine M. Coughlan ◽

Jeffrey L. Brodsky ◽

...

Keyword(s):

Point Mutations ◽

Hydrophobic Core ◽

Cellular Processes ◽

Growth Defects ◽

C Terminus ◽

Complete Breakdown ◽

Transmembrane Sequence ◽

The Stability ◽

Anchor Sequence ◽

And Function

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.

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The interplay of the type II TA system with other processes

10.7287/peerj.preprints.27770 ◽

2019 ◽

Author(s):

Wei Wen-ping ◽

Jia Wan Zhong ◽

Yang Min

Keyword(s):

Mycobacterium Tuberculosis ◽

Environmental Stress ◽

Regulatory Mechanism ◽

Phage Infection ◽

Type Ii ◽

System Research ◽

Cellular Processes ◽

The Stability ◽

And Function ◽

Insight Into

The type II toxin antitoxin (TA) system is the most well-studied TA system and is widely distributed in bacteria, especially pathogens such as Mycobacterium tuberculosis. Type II TA system plays an important role in many cellular processes, including maintaining the stability of mobile genetic elements, and bacterial altruistic suicide in response to nutritional starvation, environmental stress and phage infection. Interactions between toxin proteins and antitoxin proteins are critical for the regulation and function of type II TA systems; indeed, the understanding of their function is mainly derived from interaction and regulation of paired TA system proteins. Nonetheless, investigating interaction between unpaired TA system proteins, and the interaction between TA system proteins and other functional proteins, are becoming more common and have provided new insight into the complexity of its regulatory mechanism. In this review, we outlined the cross-interaction between TA system proteins, and the interaction between TA system proteins and other functional proteins, and we are trying to explain novel mechanism of TA system in the regulation of cellular activities. On this basis, we further discussed the knowledge and physiological implications of the relevant aspects of TA system research.

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The interplay of the type II TA system with other processes

10.7287/peerj.preprints.27770v1 ◽

2019 ◽

Author(s):

Yang Min ◽

Wei Wen-ping ◽

Jia Wan Zhong ◽

He ZhengGuo

Keyword(s):

Mycobacterium Tuberculosis ◽

Environmental Stress ◽

Regulatory Mechanism ◽

Phage Infection ◽

Type Ii ◽

System Research ◽

Cellular Processes ◽

The Stability ◽

And Function ◽

Insight Into

The type II toxin antitoxin (TA) system is the most well-studied TA system and is widely distributed in bacteria, especially pathogens such as Mycobacterium tuberculosis. Type II TA system plays an important role in many cellular processes, including maintaining the stability of mobile genetic elements, and bacterial altruistic suicide in response to nutritional starvation, environmental stress and phage infection. Interactions between toxin proteins and antitoxin proteins are critical for the regulation and function of type II TA systems; indeed, the understanding of their function is mainly derived from interaction and regulation of paired TA system proteins. Nonetheless, investigating interaction between unpaired TA system proteins, and the interaction between TA system proteins and other functional proteins, are becoming more common and have provided new insight into the complexity of its regulatory mechanism. In this review, we outlined the cross-interaction between TA system proteins, and the interaction between TA system proteins and other functional proteins, and we are trying to explain novel mechanism of TA system in the regulation of cellular activities. On this basis, we further discussed the knowledge and physiological implications of the relevant aspects of TA system research.

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mRNA Post-Transcriptional Regulation by AU-Rich Element-Binding Proteins in Liver Inflammation and Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21186648 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6648

Author(s):

Dobrochna Dolicka ◽

Cyril Sobolewski ◽

Marta Correia de Sousa ◽

Monika Gjorgjieva ◽

Michelangelo Foti

Keyword(s):

Liver Diseases ◽

Binding Proteins ◽

Current Knowledge ◽

Cellular Processes ◽

Complex Processes ◽

Cellular Context ◽

Inflammatory Processes ◽

The Stability ◽

And Function ◽

Post Transcriptional Regulation

AU-rich element-binding proteins (AUBPs) represent important post-transcriptional regulators of gene expression. AUBPs can bind to the AU-rich elements present in the 3’-UTR of more than 8% of all mRNAs and are thereby able to control the stability and/or translation of numerous target mRNAs. The regulation of the stability and the translation of mRNA transcripts by AUBPs are highly complex processes that occur through multiple mechanisms depending on the cell type and the cellular context. While AUBPs have been shown to be involved in inflammatory processes and the development of various cancers, their important role and function in the development of chronic metabolic and inflammatory fatty liver diseases (FLDs), as well as in the progression of these disorders toward cancers such as hepatocellular carcinoma (HCC), has recently started to emerge. Alterations of either the expression or activity of AUBPs are indeed significantly associated with FLDs and HCC, and accumulating evidence indicates that several AUBPs are deeply involved in a significant number of cellular processes governing hepatic metabolic disorders, inflammation, fibrosis, and carcinogenesis. Herein, we discuss our current knowledge of the roles and functions of AUBPs in liver diseases and cancer. The relevance of AUBPs as potential biomarkers for different stages of FLD and HCC, or as therapeutic targets for these diseases, are also highlighted.

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The Actin-Bundling Protein L-Plastin: A Critical Regulator of Immune Cell Function

International Journal of Cell Biology ◽

10.1155/2012/935173 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 44

Author(s):

Sharon Celeste Morley

Keyword(s):

B Lymphocyte ◽

Cell Function ◽

Immune Cell ◽

Specific Protein ◽

Cellular Processes ◽

Cell Functions ◽

Antigen Receptor Signaling ◽

Cross Links ◽

The Stability ◽

And Function

L-plastin is a leukocyte-specific protein that cross-links actin filaments into tight bundles, increasing the stability of actin-based structures such as podosomes and lamellipodia. While first identified as an abundant cytoplasmic protein in hematopoietically derived cells over 25 years ago, the requirement for L-plastin in multiple functions critical for immunity, such as antigen receptor signaling, adhesion, and motility, has only recently become clear. L-plastin has been identified as an important component in cellular processes critical for neutrophil, macrophage, osteoclast, eosinophil, and T- and B-lymphocyte biology. Following a brief description of the structure and function of L-plastin, the regulation of immune cell functions by L-plastin will be reviewed in detail.

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Characteristics and Function of the Chitin Binding Protein from Xenorhabdus nematophila

Protein and Peptide Letters ◽

10.2174/0929866526666190327143335 ◽

2019 ◽

Vol 26 (6) ◽

pp. 414-422

Author(s):

Jia Liu ◽

Ping Song ◽

Jie Zhang ◽

Ziyan Nangong ◽

Xiaobei Liu ◽

...

Keyword(s):

Antifungal Activity ◽

Growth Inhibition ◽

Insecticidal Activity ◽

Spore Germination ◽

Binding Protein ◽

Colloidal Chitin ◽

Inhibition Rate ◽

Xenorhabdus Nematophila ◽

Chitin Binding Protein ◽

And Function

Background: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). Objective: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. Methods: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. Results: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. Conclusion: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

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