SnapShot: Cartography of Intramembrane Proteolysis.

Intramembrane proteases hydrolyze peptide bonds within the cell membrane as the decision-making step of various signaling pathways or during general proteostasis. Although initially thought to be rare, fourteen proteases from four superfamilies are now known to be distributed among nearly every membrane compartment of a human cell. Each protease is endowed with specific enzymatic properties that determine both substrate choice and outcome.

Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity.

Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K(d) ~190 µM/0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k(cat) values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications.

Crystal Structures and Inhibition Kinetics Reveal a Two-Stage Catalytic Mechanism with Drug Design Implications for Rhomboid Proteolysis.

Intramembrane proteases signal by releasing proteins from the membrane, but despite their importance, their enzymatic mechanisms remain obscure. We probed rhomboid proteases with reversible, mechanism-based inhibitors that allow precise kinetic analysis and faithfully mimic the transition state structurally. Unexpectedly, inhibition by peptide aldehydes is non-competitive, revealing that in the Michaelis complex, substrate does not contact the catalytic center. Structural analysis in a membrane revealed that all extracellular loops of rhomboid make stabilizing interactions with substrate, but mainly through backbone interactions, explaining rhomboid's broad sequence selectivity. At the catalytic site, the tetrahedral intermediate lies covalently attached to the catalytic serine alone, with the oxyanion stabilized by unusual tripartite interactions with the side chains of H150, N154, and the backbone of S201. We also visualized unexpected substrate-enzyme interactions at the non-essential P2/P3 residues. These "extra" interactions foster potent rhomboid inhibition in living cells, thereby opening avenues for rational design of selective rhomboid inhibitors.

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 ß-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.

Cys-labeling kinetics of membrane protein GlpG: a role for specific SDS binding and micelle changes?

Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MFSDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MFSDS range, along with exchange from the native state at low MFSDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MFSDS at 0-0.2 MFSDS (i.e., native conditions). Thus, an increase in MFSDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MFSDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.

Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating.

Intramembrane proteases catalyse the signal-generating step of various cell signalling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration. Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, here we focus on intramembrane proteases containing domains known to exert regulatory functions in other contexts, and characterize a rhomboid protease that harbours calcium-binding EF-hands. We find calcium potently stimulates proteolysis by endogenous rhomboid-4 in Drosophila cells, and, remarkably, when rhomboid-4 is purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activates proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediate calcium stimulation. Rhomboid regulation is not orchestrated by either dimerization or substrate interactions. Instead, calcium increases catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane can be cleaved but lose the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying rhomboid functions by investigating upstream inputs that trigger proteolysis.

A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP.

Hypoxic growth of fungi requires sterol regulatory element-binding protein (SREBP) transcription factors, and human opportunistic fungal pathogens require SREBP activation for virulence. Proteolytic release of fission yeast SREBPs from the membrane in response to low oxygen requires the Golgi membrane-anchored Dsc E3 ligase complex. Using genetic interaction arrays, we identified Rbd2 as a rhomboid family protease required for SREBP proteolytic processing. Rbd2 is an active, Golgi-localized protease that cleaves the transmembrane segment of the TatA rhomboid model substrate. Epistasis analysis revealed that the Dsc E3 ligase acts on SREBP prior to cleavage by Rbd2. Using APEX2 proximity biotinylation, we demonstrated that Rbd2 binds the AAA-ATPase Cdc48 through a C-terminal SHP box. Interestingly, SREBP cleavage required Rbd2 binding of Cdc48, consistent with Cdc48 acting to recruit ubiquitinylated substrates. In support of this claim, overexpressing a Cdc48-binding mutant of Rbd2 bypassed the Cdc48 requirement for SREBP cleavage, demonstrating that Cdc48 likely plays a role in SREBP recognition. In the absence of functional Rbd2, SREBP precursor is degraded by the proteasome, indicating that Rbd2 activity controls the balance between SREBP activation and degradation.

Single-Molecule Analyses Reveal Rhomboid Proteins Are Strict and Functional Monomers in the Membrane.

Intramembrane proteases hydrolyze peptide bonds within the membrane as a regulatory paradigm that is conserved across all forms of cellular life. Many of these enzymes are thought to be oligomeric, and that their resulting quaternary interactions form the basis of their regulation. However, technical limitations have precluded directly determining the oligomeric state of intramembrane proteases in any membrane. Using single-molecule photobleaching, we determined the quaternary structure of 10 different rhomboid proteins (the largest superfamily of intramembrane proteases) and six unrelated control proteins in parallel detergent micelle, planar supported lipid bilayer, and whole-cell systems. Bacterial, parasitic, insect, and human rhomboid proteases and inactive rhomboid pseudoproteases all proved to be monomeric in all membrane conditions but dimeric in detergent micelles. These analyses establish that rhomboid proteins are, as a strict family rule, structurally and functionally monomeric by nature and that rhomboid dimers are unphysiological.

A guide to the rhomboid protein superfamily in development and disease.

Rhomboid proteins are considered to be the most widespread membrane proteins across all forms of life. This superfamily comprises both active intramembrane serine proteases that catalyze the release of factors from the membrane, and a eukaryotic subset of non-catalytic members in which rhomboid architecture supports deviating functions. Although rhomboid was discovered in genetic studies of insect development, rhomboid research has broadened dramatically over the past 15 years; rhomboid enzymes are now the best biophysically understood of all intramembrane proteases, and are considered promising therapeutic targets for diseases ranging from parasitic infections to Parkinsonian neurodegeneration. Perhaps the most rapid progress has come with the catalytically inert rhomboid proteins, some of which regulate protein trafficking and/or function, and their prominence is underscored by clinical mutations. Such a diverse collection of advances mark an excellent point to review the state of this vibrant area of research, not because central questions have been answered, but instead because a firm grip in key areas has been established, and the field is now poised for breakthroughs.

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops.

Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2-3 residues throughout the 178-residue transmembrane domain) of GlpG, a membrane-embedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3-6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denatured-state-level structure in the transition state. We propose that loops 1-3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated.

A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells.

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory.

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 facilitates host-cell invasion but is dispensable for replication.

Apical membrane antigen 1 (AMA1) is a conserved transmembrane adhesin of apicomplexan parasites that plays an important role in host-cell invasion. Toxoplasma gondii AMA1 (TgAMA1) is secreted onto the parasite surface and subsequently released by proteolytic cleavage within its transmembrane domain. To elucidate the function of TgAMA1 intramembrane proteolysis, we used a heterologous cleavage assay to characterize the determinants within the TgAMA1 transmembrane domain (ALIAGLAVGGVLLLALLGGGCYFA) that govern its processing. Quantitative analysis revealed that the TgAMA1(L/G) mutation enhanced cleavage by 13-fold compared with wild type. In contrast, the TgAMA1(AG/FF) mutation reduced cleavage by 30-fold, whereas the TgAMA1(GG/FF) mutation had a minor effect on proteolysis; mutating both motifs in a quadruple mutant blocked cleavage completely. We then complemented a TgAMA1 conditional knockout parasite line with plasmids expressing these TgAMA1 variants. Contrary to expectation, variants that increased or decreased TgAMA1 processing by >10-fold had no phenotypic consequences, revealing that the levels of rhomboid proteolysis in parasites are not delicately balanced. Only parasites transgenically expressing or carrying a true knock-in allele of the uncleavable TgAMA1(AG/FF+GG/FF) mutant showed a growth defect, which resulted from inhibiting invasion without perturbing intracellular replication. These data demonstrate that TgAMA1 cleavage plays a role in invasion, but refute a recently proposed model in which parasite replication within the host cell is regulated by intramembrane proteolysis of TgAMA1.

Mechanisms and cellular functions of intramembrane proteases.

The turn of the millennium coincided with the branding of a fundamentally different class of enzyme - proteases that reside immersed inside the membrane. This new field was the convergence of completely separate lines of research focused on cholesterol homeostasis, Alzheimer's disease, and developmental genetics. None intended their ultimate path, but soon became a richly-integrated fabric for an entirely new field: regulated intramembrane proteolysis. Our aim in this Special Issue is to focus on the ancient and nearly ubiquitous enzymes that catalyze this unexpected yet important reaction. The pace of progress has been dramatic, resulting in a rapidly-expanding universe of known cellular functions, and a paradigm shift in the biochemical understanding of these once heretical enzymes. More recently, the first therapeutic successes have been attained by targeting an intramembrane protease. We consider these advances and identify oncoming opportunities in four parts: growing spectra of cellular roles, insights into biochemical mechanisms, therapeutic strategies, and newly-emerging topics. Recent studies also expose challenges for the future, including non-linear relationships between substrate identification and physiological functions, and the need for potent and specific, not broad-class, inhibitors.

Architectural and thermodynamic principles underlying intramembrane protease function.

Intramembrane proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting intramembrane proteolysis is an essential but unattained goal. We integrated new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium unfolding and refolding and quantitative protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid proteases maintain low intrinsic thermodynamic stability (ΔG = 2.1-4.5 kcal mol(-1)) resulting from a multitude of generally weak transmembrane packing interactions, making them highly responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive 'heat map' of the physiochemical anatomy underlying membrane-immersed enzyme function at, what is to our knowledge, unprecedented resolution.

Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry.

Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix alpha4 approximately 10 A below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix alpha5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix alpha5.

Core principles of intramembrane proteolysis: comparison of rhomboid and site-2 family proteases.

Cleavage of proteins within their membrane-spanning segments is an ancient regulatory mechanism that has evolved to control a myriad of cellular processes in all forms of life. Although three mechanistic families of enzymes have been discovered that catalyze hydrolysis within the water-excluding environment of the membrane, how they achieve this improbable reaction has been both a point of controversy and skepticism. The crystal structures of rhomboid and site-2 protease, two different classes of intramembrane proteases, have been solved recently. Combined with current biochemical analyses, this advance provides an unprecedented view of how nature has solved the problem of facilitating hydrolysis within membranes in two independent instances. We focus on detailing the similarities between these unrelated enzymes to define core biochemical principles that govern this conserved regulatory mechanism.

Taking the plunge: integrating structural, enzymatic and computational insights into a unified model for membrane-immersed rhomboid proteolysis.

Rhomboid proteases are a fascinating class of enzymes that combine a serine protease active site within the core of an integral membrane protein. Despite having key roles in animal cell signalling and microbial pathogenesis, the membrane-immersed nature of these enzymes had long imposed obstacles to elucidating their biochemical mechanisms. But recent multidisciplinary approaches, including eight crystal structures, four computer simulations and nearly 100 engineered mutants interrogated in vivo and in vitro, are coalescing into an integrated model for one rhomboid orthologue in particular, bacterial GlpG. The protein creates a central hydrated microenvironment immersed below the membrane surface to support hydrolysis by its serine protease-like catalytic apparatus. Four conserved architectural elements in particular act as 'keystones' to stabilize this structure, and the lateral membrane-embedded L1 loop functions as a 'flotation device' to position the protease tilted in the membrane. Complex interplay between lateral substrate gating by rhomboid, substrate unwinding and local membrane thinning leads to intramembrane proteolysis of selected target proteins. Although far from complete, studies with GlpG currently offer the best prospect for achieving a thorough and sophisticated understanding of a simplified intramembrane protease.