DEFINITION
Melusin is a 38KDa muscle-specific chaperone protein playing a pivotal role in heart protection from stress conditions. This activity is fulfilled via activation of selective biochemical signalling pathway, thus triggering cardiomyocyte hypertrophy and protecting cells from apoptosis.
Melusin is a member of the CHORD-containing proteins family which in mammals has two known members: Melusin and Chp-1 .
These two proteins evolved from a common single ancestor, which can be found in vertebrates like C.Elegans and D. Melanogaster, after gene duplication.
The vertebrate ancestor of Melusin and Chp-1 was in turn generated from the fusion of two ancient genes, as demonstrated by the fact that plants this process did not happen and the two moieties are still expressed as different molecules: Rar1 and Sgt1. Both these proteins are involved in plant resistance to pathogen infection.
The formation of Melusin ancestor gene is an example of the rosetta stone principle, which states that when two proteins functionally interacts, they tend to form a single polypeptide during evolution, so as to improve their coordinated activity.
THE GENE
In human Melusin gene is localized on the X chromosome (location: Xq12.1–13 ), in a region of 3637 bp on the forward strand. The mRNA encodes for a 347aa polypeptide made up of 11 exons
Official Symbol: ITGB1BP2 and Name: integrin beta 1 binding protein (melusin) 2 [Homo sapiens]
Other Aliases: MSTP015, CHORDC3, ITGB1BP, MELUSIN, MGC119214
Other Designations: OTTHUMP00000023501; integrin beta-1-binding protein 2
CHEMICAL STRUCTURE AND IMAGES
Melusin is characterized by two N-terminal CHORD (Cysteine and Histidine rich) domains, followed by a C-terminal CS (CHORD and Sgt1) domain, and finally by a short acidic mojety.
The CHORD domains contain a series of cysteine and hystidine residues spaced according to a pattern that was well conserved during evolution. These residues involved in the coordination of 5 Zinc ions , which are extremely important for the correct three-dimensional folding of the CHORD domains.
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The CS domain is typical of co-chaperone proteins like the p23.
The C-terminal acidic region is characterized by the presence of many residues of aspartic and glutamic adic, that are able to bind Calcium at low affinity, but at high avidity.
No information is available for Melusin three-dimensional structure, yet there are models for both the CHORD and the CS domain that were obtained from Rar1 and Sgt1.
Rar1-CHORD is a metallo-protein domain with two structural Zn2+ ions. The CHORD domain has a cylindrical structure ∼54 Å long and ∼13 Å in diameter. The C-terminal lobe consists of a three-stranded antiparallel β sheet with one face covered by a short α helix. The N-terminal lobe is devoid of secondary structure, apart from a short β strand, which makes an antiparallel interaction with a β strand extending from the C-terminal lobe, crossing back onto the N-terminal lobe.
The CHORD domain of Rar1 is involved in two interactions: one with Hsp90 , the other with Rar1.
The modeled structure of the CS domain is a β-sandwich made of seven antiparallel β-strands, with β-strands 1, 2, 6, and 7 on one face and β-strands 3, 4, and 5 forming the other face. Two α-turns, α1 and α2, precede β-strands 3 and 6, respectively.
The double-face of the CS domain of Sgt1 is important for its function, as it allows the simultaneously bind Hsp90 and RarI.
As the two regions of Hsp90 bound by Rar1 and Sgt1 are different, the three molecules form a exameric complex.
Since Melusin is highly homologue to Rar1 and Sgt1, these structural informations are likely to be valid also for Melusin.
Indeed, Melusin can bind Hsp90 via its CHORD. However, the CS domain of Melusin has lost the ability to interact with Hsp90.
Protein Aminoacids Percentage
Compared with the median amino acid percentage in vertebrates , Melusin is not dramatically different. However, there are some remarkable characteristics.
First, there is a higher abundance of glutamic acid. This determinates a low predicted isoelectric point of 5,1 and a high negative charge at neutral ph of about 17 . Taking into account that Melusin is predicted to bind 5 Zn++ ions, the net charge would be of minus 7. This negative charge justifies the ability of Melusin to bind further metal ions, namely Ca++ as aforementioned. This, in turn, suggest that Melusin could work as a sensor of muscle contraction, by modulating some activity according to the calcium intracellular concentration
Secondly, there are increased contents of proline and leucine, which are both apolar amino acid. This could be important for the chaperone activity of Melusin, as most chaperone clients expose apolar regions that must be bound in order to avoid protein unfolding and precipitation.
Moreover, a high proline content is consistent with the expression of Melusin in non-proliferating and collagen-syntetizing cells .
Third, histidine and cysteine are slightly more abundant, as predicted from the presence of the two CHORD domains.
Finally, there is less lysine and threonine than those present on average.
When the two main domains of Melusin, the CHORD and the CS regions are analyzed , it is interesting to notice that cysteine is exclusively present in the former mojety, while the latter one is particularly rich in glutamic acid, as described earlier.
Another observation is the evident enrichment of leucine in the CHORD domain, which indicates that Melusin can be sintetyzed when there is a strong proteic syntesis (as leucine is a well-known inducer of mTOR). On the other hand, in the CS domain the glutamic acid/glutamine ratio is biased towards the first aminoacid, pointing at Melusin being syntetized more efficiently when the oxidative metabolism of the cell is good.
These two "signals" present in the two domains of Melusin (that fused during evolution) are evident only when the ammioacidic analysis is performed separately.
Another interesting analysis is the comparison of Melusin with its close homologue Chp-1. As they evolved from the same ancestor, yet their expression patterns and functions are extremely different, it is expected that a difference in the aminoacid composition would reflect this evolutionary diversification.
Interestingly, Chp-1 is more rich of lysine, which is consistent with the expression in proliferating cells (as lysine is indispensable for a strong hystone biosintesys).
SYNTHESIS AND TURNOVER
REGULATION OF mRNA AND PROTEIN SYNTESIS
MUSCLE DEVELOPMENT
Melusin is expressed selectively in skeletal and myocardial muscle
Moreover, Melusin expression is regulated during muscle differentiation. This was evidenced by the analysis of the C2C12 myogenic cell line, which can be induced to differentiate to form myotubes by serum starvation. Melusin expression was tested both by Western and Northern blotting. Melusin was absent in undifferentiated myoblasts, and its expression was turned on in differentiated myotubes after 6 days of serum starvation.
Melusin expression was also examined during mouse embryonic developmentin vivo. Melusin protein and mRNA became detectable in embryo limbs at day 15 (E15), reached a maximum in newborn mice, and declined in adult limb muscles. During heart development, on the other hand, Melusin level remains steady with no major changes in expression from embryonic day 15 to adult stage.
To investigate if Melusin expression is regulated in regenerating adult muscle, experiments were performed to induce regeneration of mouse tibialis anterior following freeze injury. 3, 6, 9, and 12 days after freeze trauma, muscles were collected and Melusin expression was investigated by Western blot analysis on total protein extracts using normal muscle as control. Melusin is up-regulated from day 6 on during muscle regeneration, consistent with a role of this molecule in myogenetic processes.
STRESS-RESPONSE
The analysis of Melusin transgenic mouse models by gene expression profiling evidenced that Melusin is co-regulated with heat-shock proteins Hsp90 and Hsp70 This co-regulation is true both at a trascriptional and translational level.
Heat shock protein expression is enhanced by multiple stress stimuli, including heat shock and mechanical load of the myocardium. Therefore, it was investigated whether Melusin expression was up-regulated by these stimuli by subjecting mice to either in vivo heat shock or to mechanical load. While Hsp70 and Hsp90 were strongly up-regulated in heart 10 and 30 h after heat shock, Melusin expression was not significantly modified. Similar results were obtained analyzing skeletal muscles (tibialis anterior, gastrocnemius and quadriceps) at the same time points. On the other hand when mechanical load was imposed to the left ventricle walls, both heat shock proteins like Hsp70 and Hsp90 and melusin were significantly up-regulated. This suggests that Melusin is a stress-response protein, whose transcription is partially co-regulated with chaperons, yet specifically responsive to mechanical stress
Moreover, when the expression of Melusin is analyzed at later time points after the imposition of pressure overload, it is evidend that it significantly correlates with heart function as monitored by echocardiography. In fact, during the initial compensatory hypertrophic phase, Melusin is up-regulated, but its levels return comparable to basal when heart failure is evident .
OTHER SUGGESTIONS
As Melusin tertiary structure requires zinc, as described earlier, dietary intake of Zinc may strongly affect Melusin biosintesys at a post-transcriptional event.
Moreover, Zinc is a foundamental cofactor of carbonic anhydrase, therefore boosting the Krebs cycle. Zinc is also present in the secretion vescicles containing insulin . Therefore, this metal ion can be part of a signalling module that increases glucose uptake and utilization.
Melusin expression could therefore be linked to such a situation of high-energy in the cell.
POST TRANSLATIONAL MODIFICATIONS AND DEGRATATION
Up to date, there are no direct evidence of any post-traslational modification of Melusin, nor it is known any particular mechanism involved in its degradation.
CELLULAR FUNCTIONS
CELLULAR LOCALIZATION
In scheletal muscle, Melusin localizes at costameres
As Melusin was first identified as interactor of the beta1d integrin cytoplasmic domain ,its sub-cellular localization is predicted to be, at least partiallly, immediatly close to the cell-membrane, as part of the complex of proteins involved in the attachment of the cell to the extracellular matrix.
ENZYMATIC ACTIVITY
Melusin is devoted of any domain involved in a real enzymatic activity. However, it can work as a chaperone protein , protecting stressed proteins from unfolding and aggregation
BIOLOGICAL FUNCTION
IN VIVO ACTIVITY: REGULATION OF HEART HYPERTROPHY
Using loss and gain of function genetically modified mouse models it was demonstrated that Melusin is required to trigger cardiomyocyte hypertrophy in response to stress stimuli such as mechanical overload. Lack of melusin leads to reduced left ventricle hypertrophy and accelerates the evolution toward heart dilation in response to pressure overload . On the other hand forced melusin expression in heart allows the development of sustained concentric hypertrophy and prevents the evolution toward heart failure .
Progression to cardiac failure is related to the intricate balance between the activation of protective and deleterious signaling pathways . In this regard, Melusin is a molecule that is involved in a hypertrophic signaling pathway and is also protective against the development of heart failure.
CELLULAR ACTIVITIES: REGULATION OF CARDIOMYOCYTE HYPERTROPHY AND APOPTOSIS
Cultured cardiomyocytes from Melusin overexpressing mice show enhanced hypertrophy and protection from apoptosis induced by oxidative stress.
BIOCHEMICAL ACTIVITIES: ACTIVATION OF ERK1/2 AND AKT.
Melusin is necessary for the proper activation of ERK1/2 and AKT downstream of integrins in cardiomyocytes.These molecules are important for the development of cell hypertrophy and protection from apoptosis
INTERACTIONS
Melusin can directly interact with the cytoplasmic domain of integrins and with N-terminal of the molecular chaperone Hsp90
Moreover, Melusin can interact with the MAPK scaffold IQGAP1 , and the three members of the biochemical cascade: Raf-1 , MEK1/2 and ERK1/2
It was proposed that Melusin can activate this signalling cascade via its chaperone acivity per se as well as via recruitment of Hsp90
REGULATION
Currently, no mechanism is known that regulates Melusin function.
As Melusin can bind calcium with its C-terminal acidic tail, as described above, it is possible that it could work as a sensor of Muscle contraction, adjusting its activity according to the intracellular concentration of calcium
Another consideration is that Melusin have many cysteine residues in its CHORD domains, that are possibly undergoing disulfide bond formation. Therefore, Melusin could be a sensor of the redox condition of a cell.
DIAGNOSTIC USE
There is no actual diagnostic use of Melusin.
It had been analyzed the presence of nucleotide variations of Melusin in hypertensive and cardiopatic patients , but only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11.
The three variations of the ITGB1BP2 gene have been detected in families of patients affected either by hypertension or primary hypertrophic cardiomyopathy; however, a clear genotype/phenotype correlation was not evident. Functional results and bioinformatic analysis excluded a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.
This analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.
THERAPEUTIC USE
Melusin is protected by an international patent for its usage as therapy for heart failure, but presently there are no estabilished therapies available.
