Rhomboid protease
Encyclopedia
The rhomboid proteases are a family of enzyme
s that exist in almost all species. They are proteases: they cut the polypeptide chain of other proteins. This proteolytic cleavage is irreversible in cells
, and an important type of cellular regulation. Although proteases are one of the earliest and best studied class of enzyme, rhomboids belong to a much more recently discovered type: the intramembrane protease
s. What is unique about intramembrane proteases is that their active sites are buried in the lipid bilayer
of cell membranes, and they cleave other transmembrane proteins within their transmembrane domains . About 30% of all proteins have transmembrane domains, and their regulated processing often has major biological consequences. Accordingly, rhomboids regulate many important cellular processes, and may be involved in a wide range of human diseases.
and metallo-proteases
, respectively. The presenilins and signal peptide peptidase
-like family, which are intramembrane aspartyl proteases, cleave substrates that include the Notch
receptor and the amyloid precursor protein
, which is implicated in Alzheimer’s disease. The site-2 protease family
, which are intramembrane metalloproteases, regulate among other things cholesterol
biosynthesis and stress responses in bacteria
. The different intramembrane protease families are evolutionarily and mechanistically unrelated, but there are clear common functional themes that link them. Rhomboids are perhaps the best characterised class.
, discovered in a famous genetic screen that led to a Nobel Prize
for Christiane Nüsslein-Volhard
and Eric Wieschaus . In that screen they found a number of mutants with similar phenotypes: ‘pointy’ embryonic head skeletons. They named them each with a pointy-themed name – one was rhomboid. Genetic analysis later proved that this group of genes were members of the epidermal growth factor (EGF) receptor
signalling pathway , and that rhomboid was needed to generate the signal that activates the EGF receptor . The molecular function of rhomboid took a bit longer to unravel but a combination of genetics and molecular techniques led to the discovery that Drosophila rhomboid and other members of the family were the first known intramembrane serine proteases .
Spitz, from its transmembrane precursor, rhomboid triggers signalling . Since then, many other important biological functions have been proposed .
was solved . These structures confirmed predictions that rhomboids have a core six transmembrane domain structure, and that the catalytic site depends on a serine and histidine catalytic dyad. The structure also explained how a proteolytic reaction, which requires water molecules, can occur in the hydrophobic environment of a lipid bilayer: one of the central mysteries of intramembrane proteases . The catalytic site was shown to be a hydrophilic indentation, protected from the lipid bilayer by surrounding transmembrane domains .
One area of uncertainty is the route by which substrates get access to the rhomboid acive site. Although substrates were initially proposed to enter between transmembrane domains 1 and 3 , evidence now strongly supports an alternative access point, between transmembrane domains 2 and 5 .
Initial work indicated that rhomboids recognise instability of the transmembrane alpha-helix as the main substrate determinant . More recently a primary sequence motif in or immediately adjacent to the transmembrane domain has been shown to be the cardinal recognition determinant of a variety of rhomboid substrates . This recognition motif directs where the substrate is cleaved. This can occur either within the transmembrane domain or just outside the membrane. Only in the former case are helix destabilising residues also necessary. Although the structure of a rhomboid/substrate complex has not yet been solved, a recent structure of the enzyme in complex with a mechanism-based inhibitor is consistent with the current understanding of rhomboid specificity,
, parasite infection, and diabetes . It is important to note, however, that there is no case yet established where a precise medical significance is fully validated.
No drugs that modulate rhomboid activity have yet been reported, although a recent study has identified small molecule, mechanism-based inhibitors that could provide a basis for future drug development .
and RHBDF2
). iRhoms can promote the ER associated degradation
(ERAD) of EGF receptor ligands . In Drosophila this provides a mechanism for regulating EGF receptor activity in the brain. This unexpected mechanism implies that the fundamental cellular quality control mechanism is exploited by multicellular organisms to regulate signalling between cells.
Phylogenetic analysis
indicates that rhomboids are in fact members of a larger rhomboid-like superfamily or clan, which includes the derlin proteins, also involved in ERAD .
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
s that exist in almost all species. They are proteases: they cut the polypeptide chain of other proteins. This proteolytic cleavage is irreversible in cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
, and an important type of cellular regulation. Although proteases are one of the earliest and best studied class of enzyme, rhomboids belong to a much more recently discovered type: the intramembrane protease
Intramembrane protease
Intramembrane proteases are enzymes that have the property of cleaving transmembrane domains of integral membrane proteins. All known intramembrane proteases are themselves integral membrane proteins with multiple transmembrane domains, and they have their active sites buried within the lipid...
s. What is unique about intramembrane proteases is that their active sites are buried in the lipid bilayer
Lipid bilayer
The lipid bilayer is a thin membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around cells. The cell membrane of almost all living organisms and many viruses are made of a lipid bilayer, as are the membranes surrounding the cell nucleus...
of cell membranes, and they cleave other transmembrane proteins within their transmembrane domains . About 30% of all proteins have transmembrane domains, and their regulated processing often has major biological consequences. Accordingly, rhomboids regulate many important cellular processes, and may be involved in a wide range of human diseases.
Intramembrane proteases
Rhomboids are intramembrane serine proteases . The other types of intramembrane protease are aspartyl-Aspartate protease
Aspartic proteases are a family of protease enzymes that use an aspartate residue for catalysis of their peptide substrates. In general, they have two highly-conserved aspartates in the active site and are optimally active at acidic pH...
and metallo-proteases
Metalloproteinase
Metalloproteinases constitute a family of enzymes from the group of proteases, classified by the nature of the most prominent functional group in their active site. These are proteolytic enzymes whose catalytic mechanism involves a metal. Most metalloproteases are zinc-dependent, but some use...
, respectively. The presenilins and signal peptide peptidase
Signal Peptide Peptidase
The Signal Peptide Peptidase is an intramembrane aspartyl protease with the conserved active site motifs 'YD' and 'GxGD' in in adjacent transmembrane domains . Its sequences is highly conserved in different vertebrate species...
-like family, which are intramembrane aspartyl proteases, cleave substrates that include the Notch
Notch
Notch may refer to:* The nock of an arrow* Notch , a Hip hop, R&B, reggae, dancehall and reggaeton artist* Notch signaling pathway, a cell signaling system present in most multicellular organisms...
receptor and the amyloid precursor protein
Amyloid precursor protein
Amyloid precursor protein is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, neural plasticity and iron export...
, which is implicated in Alzheimer’s disease. The site-2 protease family
Membrane-bound transcription factor peptidase, site 2
Membrane-bound transcription factor peptidase, site 2 also known as Site-2 Protease is an enzyme encoded by the gene which liberates the N-terminal fragment of sterol regulatory element binding protein transcription factors from membranes . S2P cleaves the transmembrane domain of SREPB, making...
, which are intramembrane metalloproteases, regulate among other things cholesterol
Cholesterol
Cholesterol is a complex isoprenoid. Specifically, it is a waxy steroid of fat that is produced in the liver or intestines. It is used to produce hormones and cell membranes and is transported in the blood plasma of all mammals. It is an essential structural component of mammalian cell membranes...
biosynthesis and stress responses in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
. The different intramembrane protease families are evolutionarily and mechanistically unrelated, but there are clear common functional themes that link them. Rhomboids are perhaps the best characterised class.
History of rhomboid discovery
Rhomboids were first named after a mutation in the fruitfly DrosophilaDrosophila
Drosophila is a genus of small flies, belonging to the family Drosophilidae, whose members are often called "fruit flies" or more appropriately pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species to linger around overripe or rotting fruit...
, discovered in a famous genetic screen that led to a Nobel Prize
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...
for Christiane Nüsslein-Volhard
Christiane Nüsslein-Volhard
Christiane Nüsslein-Volhard is a German biologist who won the Albert Lasker Award for Basic Medical Research in 1991 and the Nobel Prize in Physiology or Medicine in 1995, together with Eric Wieschaus and Edward B...
and Eric Wieschaus . In that screen they found a number of mutants with similar phenotypes: ‘pointy’ embryonic head skeletons. They named them each with a pointy-themed name – one was rhomboid. Genetic analysis later proved that this group of genes were members of the epidermal growth factor (EGF) receptor
Epidermal growth factor receptor
The epidermal growth factor receptor is the cell-surface receptor for members of the epidermal growth factor family of extracellular protein ligands...
signalling pathway , and that rhomboid was needed to generate the signal that activates the EGF receptor . The molecular function of rhomboid took a bit longer to unravel but a combination of genetics and molecular techniques led to the discovery that Drosophila rhomboid and other members of the family were the first known intramembrane serine proteases .
Biological role of rhomboids
Rhomboids were first discovered as proteases that regulate EGF receptor signalling in Drosophila. By releasing the extracellular domain of the growth factorGrowth factor
A growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. Usually it is a protein or a steroid hormone. Growth factors are important for regulating a variety of cellular processes....
Spitz, from its transmembrane precursor, rhomboid triggers signalling . Since then, many other important biological functions have been proposed .
- Although less well established than in Drosophila, there is some evidence that rhomboids may participate in growth factor signalling in mammals, including humans . They have also been implicated in ephrinEphrinIn molecular biology, ephrins are a family of proteins that are ligands of class V receptor protein-tyrosine kinases. These receptors and their ligands have been implicated in regulating neuronal axon guidance and in patterning of the developing nervous system and may also serve a patterning and...
signalling and the cleavage of the anticoagulantAnticoagulantAn anticoagulant is a substance that prevents coagulation of blood. A group of pharmaceuticals called anticoagulants can be used in vivo as a medication for thrombotic disorders. Some anticoagulants are used in medical equipment, such as test tubes, blood transfusion bags, and renal dialysis...
protein thrombomodulinThrombomodulinThrombomodulin, CD141 or BDCA-3 is an integral membrane protein expressed on the surface of endothelial cells. In humans, thrombomodulin is encoded by the THBD gene...
. - All eukaryotes have a mitochondrial rhomboid. In yeast this has been shown to control mitochondrial function and morphology by regulating membrane fusion via the cleavage of a dynaminDynaminDynamin is a GTPase responsible for endocytosis in the eukaryotic cell. Dynamins are principally involved in the scission of newly formed vesicles from the membrane of one cellular compartment and their targeting to, and fusion with, another compartment, both at the cell surface as well as at the...
-like GTPase called Mgm1p, the orthologue of human OPA1 . In Drosophila, the mitochondrial rhomboid also regulates mitochondrial membrane fusion . In mammals too, mitochondrial function is disrupted in mutants of PARLPARLPresenilins-associated rhomboid-like protein, mitochondrial is a protein that in humans is encoded by the PARL gene . It is a member of the rhomboid family of intramembrane serine proteases .-Further reading:...
, the mitochondrial rhomboid, but the range of functions is more complex. PARL regulates the remodelling of mitochondrial cristae , is implicated in cell death and metabolismMetabolismMetabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
, and there is increasing evidence of an important role in Parkinson's DiseaseParkinson's diseaseParkinson's disease is a degenerative disorder of the central nervous system...
; - Apicomplexan parasitesApicomplexaThe Apicomplexa are a large group of protists, most of which possess a unique organelle called apicoplast and an apical complex structure involved in penetrating a host's cell. They are unicellular, spore-forming, and exclusively parasites of animals. Motile structures such as flagella or...
like PlasmodiumPlasmodiumPlasmodium is a genus of parasitic protists. Infection by these organisms is known as malaria. The genus Plasmodium was described in 1885 by Ettore Marchiafava and Angelo Celli. Currently over 200 species of this genus are recognized and new species continue to be described.Of the over 200 known...
(the agent that causes malariaMalariaMalaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
) and Toxoplasma appear to use rhomboids to cleave cell surface proteins that participate in the host invasion process ; they have also been implicated in the pathogenicity of other parasites ; - A rhomboid in the Gram-negativeGram-negativeGram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
bacterium Providencia stuartii is required for the function of the twin-arginine protein translocation (TAT) machinery .
Structure and enzyme function of rhomboids
Rhomboids were the first intramembrane proteases for which a high resolution crystal structureCrystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
was solved . These structures confirmed predictions that rhomboids have a core six transmembrane domain structure, and that the catalytic site depends on a serine and histidine catalytic dyad. The structure also explained how a proteolytic reaction, which requires water molecules, can occur in the hydrophobic environment of a lipid bilayer: one of the central mysteries of intramembrane proteases . The catalytic site was shown to be a hydrophilic indentation, protected from the lipid bilayer by surrounding transmembrane domains .
One area of uncertainty is the route by which substrates get access to the rhomboid acive site. Although substrates were initially proposed to enter between transmembrane domains 1 and 3 , evidence now strongly supports an alternative access point, between transmembrane domains 2 and 5 .
Rhomboid specificity
Rhomboids do not cleave all transmembrane domains. In fact, they are highly specific, with a limited number of substrates. All natural substrates known so far contain a single transmembrane domain; most are type 1, with their amino termini in the luminal/extracellular compartment, although one exception has been proposed: Star, a type 2 membrane protein (i.e. with opposite topology: the amino terminus is cytoplasmic) in Drosophila . The specificity of rhomboids underlies their ability to control functions in a wide range of biological processes and, in turn, understanding what makes a particular transmembrane domain into a rhomboid substrate can shed light on rhomboid function in different contexts.Initial work indicated that rhomboids recognise instability of the transmembrane alpha-helix as the main substrate determinant . More recently a primary sequence motif in or immediately adjacent to the transmembrane domain has been shown to be the cardinal recognition determinant of a variety of rhomboid substrates . This recognition motif directs where the substrate is cleaved. This can occur either within the transmembrane domain or just outside the membrane. Only in the former case are helix destabilising residues also necessary. Although the structure of a rhomboid/substrate complex has not yet been solved, a recent structure of the enzyme in complex with a mechanism-based inhibitor is consistent with the current understanding of rhomboid specificity,
Medical significance of rhomboids
The diversity of biological functions already known to depend on rhomboids is reflected in evidence that rhomboids play a role in a variety if diseases including cancerCancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
, parasite infection, and diabetes . It is important to note, however, that there is no case yet established where a precise medical significance is fully validated.
No drugs that modulate rhomboid activity have yet been reported, although a recent study has identified small molecule, mechanism-based inhibitors that could provide a basis for future drug development .
The rhomboid-like family
Rhomboid proteases appear to be conserved in all eukaryotes and the vast majority of prokaryotes. Bioinformatic analysis highlights that some members of the rhomboid family lack the amino acid residues essential for proteolysis, implying that they cannot cleave substrates. These ‘pseudoproteases’ include a subfamily that have been named the iRhoms (also known as RHBDF1RHBDF1
Inactive rhomboid protein 1 also known as rhomboid 5 homolog 1 or rhomboid family member 1 is a protein that in humans is encoded by the RHBDF1 gene. The alternative name iRhom1 has been proposed, in order to clarify that it is a catalytically inactive member of the rhomboid family of...
and RHBDF2
RHBDF2
Rhomboid family member 2 is a protein that in humans is encoded by the RHBDF2 gene . The alternative name iRhom2 has been proposed, in order to clarify that it is a catalytically inactive member of the rhomboid family of intramembrane serine proteases ....
). iRhoms can promote the ER associated degradation
ERAD
Endoplasmic-reticulum-associated protein degradation designates a cellular pathway which targets misfolded proteins of the endoplasmic reticulum for ubiquitination and subsequent degradation by a protein-degrading complex, called the proteasome....
(ERAD) of EGF receptor ligands . In Drosophila this provides a mechanism for regulating EGF receptor activity in the brain. This unexpected mechanism implies that the fundamental cellular quality control mechanism is exploited by multicellular organisms to regulate signalling between cells.
Phylogenetic analysis
Phylogenetics
In biology, phylogenetics is the study of evolutionary relatedness among groups of organisms , which is discovered through molecular sequencing data and morphological data matrices...
indicates that rhomboids are in fact members of a larger rhomboid-like superfamily or clan, which includes the derlin proteins, also involved in ERAD .
External links
- http://merops.sanger.ac.uk/cgi-bin/famsum?family=S54
- http://expasy.org/enzyme/3.4.21.105