Rhomboid Serine Proteases Research Articles

KOMPEITO, an Atypical Arabidopsis Rhomboid-Related Gene, Is Required for Callose Accumulation and Pollen Wall Development.

Fertilization is a key event for sexually reproducing plants. Pollen–stigma adhesion, which is the first step in male–female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a β-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen–stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.

Unraveling the Complexity of the Rhomboid Serine Protease 4 Family of Babesia bovis Using Bioinformatics and Experimental Studies.

Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus investigated B. bovis ROM4 coding genes; their genomic organization; their expression in in vitro cultured asexual (AS) and sexual stages (SS); and strain polymorphisms. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5. There are moderate degrees of sequence identity between them, except for rom4.3 and 4.4, which are almost identical. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4, respectively, display 18-fold and 218-fold significantly higher (p < 0.01) levels of transcription in SS than in AS, suggesting a role in gametogenesis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between the stages. ROM4 polymorphisms among geographic isolates were essentially restricted to the number of tandem repeats of a 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host–pathogen interactions and are possibly useful targets for the development of new control strategies against this pathogen.

Alternative splice variants of rhomboid proteins: Comparative analysis of database entries for select model organisms and validation of functional potential.

Background: Rhomboid serine proteases are present across many species and are often encoded in each species by more than one predicted gene. Based on protein sequence comparisons, rhomboids can be differentiated into groups - secretases, presenilin-like associated rhomboid-like (PARL) proteases, iRhoms, and "inactive" rhomboid proteins. Although these rhomboid groups are distinct, the different types can operate simultaneously. Studies in Arabidopsis showed that the number of rhomboid proteins working simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the Arabidopsis plastid rhomboid proteins At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two Arabidopsis plastid rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species. Methods: We thus conducted a comparative analysis of select databases to determine if the alternative splicing mechanism observed for the two Arabidopsis plastid rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the in silico observations, select splice variants from different groups were tested for activity using transgenic- and additive-based assays. These assays aimed to uncover evidence that the selected splice variants display capacities to influence processes like antimicrobial sensitivity. Results: A comparison of database entries of six widely used eukaryotic experimental models (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and Arabidopsis. Subsequent validation studies uncover evidence that the splice variants selected for testing displayed functionality in the different activity assays. Conclusions: The combined results support the hypothesis that alternative splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.

Alternative splice variants of rhomboid proteins: Comparative analysis of database entries for select model organisms and validation of functional potential

Background: Rhomboid serine proteases are present across many species and are often encoded in each species by more than one predicted gene. Based on protein sequence comparisons, rhomboids can be differentiated into groups - secretases, presenilin-like associated rhomboid-like (PARL) proteases, iRhoms, and “inactive” rhomboid proteins. Although these rhomboid groups are distinct, the different types can operate simultaneously. Studies in Arabidopsis showed that the number of rhomboid proteins working simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the Arabidopsis plastid rhomboid proteins At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two Arabidopsis plastid rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species. Methods: We thus conducted a comparative analysis of select databases to determine if the alternative splicing mechanism observed for the two Arabidopsis plastid rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the in silico observations, select splice variants from different groups were tested for activity using transgenic- and additive-based assays. These assays aimed to uncover evidence that the selected splice variants display capacities to influence processes like antimicrobial sensitivity. Results: A comparison of database entries of six widely used eukaryotic experimental models (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and Arabidopsis. Subsequent validation studies uncover evidence that the splice variants selected for testing displayed functionality in the different activity assays. Conclusions: The combined results support the hypothesis that alternative splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.

Alternative splice variants of rhomboid proteins: In silico analysis of database entries for select model organisms and validation of functional potential

Background: Rhomboid serine proteases are present in many species with sequenced genomes, and are often encoded in each species by more than one predicted gene. Based on protein sequence comparisons, rhomboids can be differentiated into groups - secretases, presenilin-like associated rhomboid-like (PARL) proteases, iRhoms, and "inactive" rhomboid proteins. Although these rhomboid groups are distinct, the different types can operate simultaneously. Studies in Arabidopsis showed that the number of rhomboid proteins working simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the Arabidopsis plastid rhomboid proteins At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two Arabidopsis plastid rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species. Methods: We thus conducted a multi-year analysis of databases to determine if the alternative splicing mechanism observed for the two Arabidopsis plastid rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the in silico findings, select splice variants from different groups were tested for activity using transgenic- and additive-based assays. These assays aimed to uncover evidence that the selected splice variants display capacities to influence processes like antimicrobial sensitivity. Results: The multi-year in silico assessment for six model experimental species (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and Arabidopsis. Subsequent validation studies uncover evidence that the splice variants selected for testing displayed functionality in the different activity assays. Conclusions: The combined results support the hypothesis that alternative splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.

Expression, Purification, and Enzymatic Characterization of Intramembrane Proteases.

Intramembrane proteases catalyze peptide bond hydrolysis in the lipid bilayer and play a key role in numerous cellular processes. These integral membrane enzymes consist of four classes: site-2 protease (S2P), rhomboid serine protease, Rce1-type glutamyl protease, and aspartyl protease exemplified by presenilin and signal peptide peptidase (SPP). Structural elucidation of these enzymes is important for mechanistic understanding of their functions, particularly their roles in cell signaling and debilitating diseases such as Parkinson's disease and Alzheimer's disease. In the past decade, rigorous effort has led to determination of the crystal structures of S2P from archaebacterium, rhomboid serine protease from E. coli (GlpG), and presenilin/SPP from archaebacterium (PSH). A novel method has been developed to express well-behaved human γ-secretase, which facilitated its structure determination by cryoelectron microscopy (cryo-EM). In this chapter, we will discuss the expression and purification of intramembrane proteases including human γ-secretase and describe the enzymatic activity assays for these intramembrane proteases.

The Mechanism of Rhomboid Intramembrane Protease

The activities of many membrane proteins, including transcription factors, receptors, growth factors, and transporters, are regulated by a cleavage event within their transmembrane domains. The intramembrane proteolytic reaction is catalyzed by highly specialized membrane-embedded proteases belonging to three mechanistic families. The rhomboid serine protease, first discovered by Drosophila genetics, represents a large family of functionally diverse membrane proteins that share a common core domain of six transmembrane segments. Extensive crystallographic analysis of the bacterial rhomboid GlpG has generated insight into how peptide hydrolysis occurs inside the hydrophobic environment of membrane bilayers. Here we examine the conformational change that takes place during substrate binding and catalysis based on x-ray structures of GlpG in complex with inhibitors and peptide analogs.

Characterization of three alternative splice variants associated with theArabidopsisrhomboid protein geneAt1g74130

Rhomboid serine proteases are grouped into three main types — secretases, presenilin-like associated rhomboid-like (PARL) proteases, and “inactive” rhomboid proteins. Although the three rhomboid groups are distinct, the different types are likely to operate within the same cell or compartment, such as observed in the plastids of Arabidopsis. There are four distinct plastid rhomboid genes at play in Arabidopsis plastids, two for active types (At1g25290 and At5g25752) and two for inactive forms (At1g74130 and At1g74140). The number of working plastid rhomboids is further increased by alternative splicing, as reported for At1g25290. To understand how the plastid rhomboid system works, it is necessary to identify all rhomboid forms in play. To this end, this study was designed to examine the alternative splicing activities of At1g74130, one of the two genes encoding proteolytically “inactive” plastid rhomboids. The exon mapping and DNA sequencing results obtained here indicate the presence of three prominent alternative splice variants in the At1g74130 transcript population. The dominant splice variant, L, encodes the full-length protein. The other two splice variants, M and S, produce proteins lacking sections from the carboxyl transmembrane domain region. The splice variants M and S appear to be at levels with functional potential and appear to adjust relative to each other during development and in response to changes in the level of Tic40, a component of the plastid translocon. The splice variant proteins themselves exhibit different characteristics with respect to rhomboid protein–substrate interactions. These differences were observed in bacterial co-expression pull-down assays and in yeast mitochondrial studies. When considered together, the data suggest that the alternative splicing of At1g74130 bears functional significance in Arabidopsis and is likely to be part of a mechanism for diversifying plastid rhomboid function.

Function and Characteristics of PINK1 in Mitochondria

Mutations in phosphatase and tensin homologue-induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease, a neurodegenerative disorder linked to mitochondrial dysfunction. Studies support the notion of neuroprotective roles for the PINK1, as it protects cells from damage-mediated mitochondrial dysfunction, oxidative stress, and cell apoptosis. PARL is a mitochondrial resident rhomboid serine protease, and it has been reported to mediate the cleavage of the PINK1. Interestingly, impaired mitophagy, an important autophagic quality control mechanism that clears the cells of damaged mitochondria, may also be an underlying mechanism of disease pathogenesis in patients for Parkinson's disease with the PARL mutations. Functional studies have revealed that PINK1 recruits Parkin to mitochondria to initiate the mitophagy. PINK1 is posttranslationally processed, whose level is definitely regulated in healthy steady state of mitochondria. As a consequence, PINK1 plays a pivotal role in mitochondrial healthy homeostasis.

Rhomboid Protease PARL Mediates the Mitochondrial Membrane Potential Loss-induced Cleavage of PGAM5

Regulated intramembrane proteolysis is a widely conserved mechanism for controlling diverse biological processes. Considering that proteolysis is irreversible, it must be precisely regulated in a context-dependent manner. Here, we show that phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is cleaved in its N-terminal transmembrane domain in response to mitochondrial membrane potential (ΔΨ(m)) loss. This ΔΨ(m) loss-dependent cleavage of PGAM5 was mediated by presenilin-associated rhomboid-like (PARL). PARL is a mitochondrial resident rhomboid serine protease and has recently been reported to mediate the cleavage of PINK1, a mitochondrial Ser/Thr protein kinase, in healthy mitochondria with intact ΔΨ(m). Intriguingly, we found that PARL dissociated from PINK1 and reciprocally associated with PGAM5 in response to ΔΨ(m) loss. These results suggest that PARL mediates differential cleavage of PINK1 and PGAM5 depending on the health status of mitochondria. Our data provide a prototypical example of stress-dependent regulation of PARL-mediated regulated intramembrane proteolysis.

An Internal Water-Retention Site in the Rhomboid Intramembrane Protease GlpG Ensures Catalytic Efficiency

Rhomboid proteases regulate key cellular pathways, but their biochemical mechanism including how water is made available to the membrane-immersed active site remains ambiguous. We performed four prolonged molecular dynamics simulations initiated from both gate-open and gate-closed states of Escherichia coli rhomboid GlpG in a phospholipid bilayer. GlpG was notably stable in both gating states, experiencing similar tilt and local membrane thinning, with no observable gating transitions, highlighting that gating is rate-limiting. Analysis of dynamics revealed rapid loss of crystallographic waters from the active site, but retention of a water cluster within a site formed by His141, Ser181, Ser185, and/or Gln189. Experimental interrogation of 14 engineered mutants revealed an essential role for at least Gln189 and Ser185 in catalysis with no effect on structural stability. Our studies indicate that spontaneous water supply to the intramembrane active site of rhomboid proteases is rare, but its availability for catalysis is ensured by an unanticipated active site element, the water-retention site.

Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

Molecular characterization of organellar rhomboid proteins inArabidopsis

The list of cellular processes involving rhomboid serine proteases and regulated intramembrane proteolysis (RIP) is growing, diverse, and spans different species. The latest entrants to the RIP phenomenon are the plant rhomboid proteases. Although natural rhomboid protease substrates and their corresponding processes have been identified from insects to mammals, the current knowledge on plant rhomboid proteases is limited. This study was thus aimed at revealing further details about the roles of three Arabidopsis organellar rhomboid proteases and like proteins. Globally, the patterns revealed at the transcript level suggest that the three Arabidopsis rhomboid proteins under study are linked partly to growth and development. Further evidence for a linkage to growth and development was obtained for the organellar rhomboid protein, At1g74130. This link was most evident during the early growth phases of the various tissues assessed, e.g., At1g74130 expression was greater in young, expanding leaves than in mature leaves. This expression pattern appears to correlate with the responses of the Arabidopsis At1g74130 mutant, where there were observable delays in germination and growth. These delays appear to be associated with the early stages of plastid development and can be observed as changes at the cellular level and at the level of chlorophyll content. The data reported here suggest that organellar rhomboid proteins, in particular At1g74130, may take part in growth and development.

Lipid Membrane Composition has A Dramatic Effect on the Dynamics of the GlpG Rhomboid Protease from Escherichia Coli

Intramembrane proteases cleave transmembrane substrates to liberate physiologically important molecules. The proteolytic activity of these enzymes can be significantly influenced by the composition of the lipid membrane. Here, we find that the composition of the lipid membrane has a dramatic effect on the motions of the GlpG rhomboid serine protease from Escherichia coli.In a 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer, conformational changes of two critical structural elements of the protease, the cap loop close to the active site and the regulatory loop L1, occur within 40ns of unconstrained molecular dynamics simulations. In contrast, in a 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) lipid bilayer, a conformational transition of the cap loop is observed only after ∼80ns, ∼20ns after that of loop L1. This sensitivity of the enzyme motions on the lipid membrane composition is explained by differences in how POPC and POPE lipid headgroups hydrogen bond among themselves, and with protein amino acids. Tight interactions between a lipid headgroup and the active site restrict the dynamics of the cap loop.An atomistic description of the structure and dynamics of the protease:substrate complex is a critical first step towards understanding how the protease works. Molecular dynamics simulations of GlpG together with the Spitz model substrate reveal that docking of the substrate to the enzyme involves a complex interplay of changes in the structure and dynamics of the substrate, the protease, and the surrounding lipid molecules.This research was supported in part by the National Institute of General Medical Sciences (GM-74637 to S.H.W) and an allocation of computer time from the National Science Foundation through the TeraGrid resources at TACC (Ranger).

Rip exposed: How ectodomain shedding regulates the proteolytic processing of transmembrane substrates

Regulated intramembrane proteolysis (Rip) controls a wide variety of cellular mechanisms such as cholesterol homeostasis, immune surveillance, cellular signaling, and β-amyloid formation in Alzheimer's disease (1). Rip of substrates is mediated by several families of intramembranously cleaving proteases (I-CLiPs), all of which perform the unique chemistry of hydrolysis within the hydrophobic lipid bilayer (2). There are four known families of I-CLiPs, each denoted by the protease that typifies each group: site-2 protease (S2P) metalloproteases, the γ-secretase and signal peptide peptidase (SPP) aspartyl proteases, and the rhomboid serine proteases. Rip cleavage of transmembrane substrates by S2P, SPP, and γ-secretase is preceded and regulated by an initial distinct cleavage in a process termed “ectodomain shedding” (Fig. 1). The reason ectodomain shedding is necessary in most Rip cases is not understood. An article in this issue of PNAS (3) has shed new light on this important question.

Intramembrane Proteolysis by the Rhomboid Serine Protease GlpG

Intramembrane proteases catalyze peptide bond cleavage of integral membrane protein substrates. This activity is crucial for many biological and pathological processes. Rhomboids are evolutionarily widespread intramembrane serine proteases that cleave type I or II membrane protein substrates. In addition to high-resolution structural studies of the E. coli representative GlpG, this presentation will discuss the ability of rhomboids to cleave unfolded multi-spanning membrane proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Structural basis for intramembrane proteolysis by rhomboid serine proteases

Intramembrane proteases catalyze peptide bond cleavage of integral membrane protein substrates. This activity is crucial for many biological and pathological processes. Rhomboids are evolutionarily widespread intramembrane serine proteases. Here, we present the 2.3-A-resolution crystal structure of a rhomboid from Escherichia coli. The enzyme has six transmembrane helices, five of which surround a short TM4, which starts deep within the membrane at the catalytic serine residue. Thus, the catalytic serine is in an externally exposed cavity, which provides a hydrophilic environment for proteolysis. Our results reveal a mechanism to enable water-dependent catalysis at the depth of the hydrophobic milieu of the membrane and suggest how substrates gain access to the sequestered rhomboid active site.