Protein Folding and Associated Diseases

Proteins are synthesized on ribosomes as nascent polypeptides in the lumen of the endoplasmic reticulum. The amino acid sequence of proteins that determines the secondary and tertiary structures is dictated by the nucleotide sequence of mRNA. In turn, mRNA sequences are determined by DNA sequences. Out of many conformational possibilities, only single conformation serves as active protein.

Molecular Chaperones: 

• The process of directing and targeting the folding of intermediate polypeptides to the fully folded native structures is aided by proteins known as molecular chaperones. 
• Chaperones bind reversibly to polypeptide segments and prevent their misfolding and premature aggregation. 
• This process involves energy expenditure provided by the hydrolysis of ATP. A major category of chaperones is heat-shock proteins, which are synthesized in both prokaryotic and eukaryotic cells in response to heat shock or other stresses like free radicles.
• There are many classes of heat-shock chaperones (HSP-60, HSP-70, and HSP-90) that are present in various organelles of the cell. 
• HSP-70 chaperones contain two domains: an ATPase domain and a peptide binding domain. These are useful to stabilize nascent polypeptides and also are able to reconform denatured forms of polypeptides. 
• The HSP-70 family of chaperones shows a high degree of sequence homology among various species. 
• Another chaperone, ealnexin, is a 90 kDa Ca2+-binding protein and is an integral membrane phosphoprotein of Endoplasmic Reticulum
• Calnexin checks the export of newly synthesized glycoproteins by complexing with misfolded glycoproteins that have undergone glycosylation. 
• If a protein cannot be folded into its proper conformation, chaperones assist in destruction. 

                   The process of folding is also facilitated by the ionic environment, cofactors, and enzymes. For example, folding is affected by protein disulfide isomerase, which catalyzes the formation of correct disulfide linkages, and by peptidyl prolyl isomerases, which catalyze the cis-trans isomerization of specific amino acidproline peptide bonds.

Protein Folding

Folding Associated Diseases:                   

                  Several diseases of protein folding are known that have the characteristic pathological mark of protein aggregation and deposits in and around the cells. The protein deposits are called amyloid and the disease is known as amyloidosis. Protein folding diseases, also known as conformational diseases, have many different etiologies, such as changes in the primary structure of proteins, defects in chaperones, and the inappropriate presence or influence of other proteins. A common though not invariable aspect of conformational protein diseases is that the aggregation of polypeptides is made up of β-structures. This is primarily due to a transition from α-helical structure to β-structure. Another feature is that the aggregates are resistant to normal proteolysis.

                 A dementia syndrome characterized by an insidious progressive decline in memory, cognition, behavioral stability, and independent function was described by Alois Alzheimer and is known as Alzheimer's disease. Age is an important risk factor for Alzheimer's disease; it affects 10% of persons over 65 years of age and about 40% of those over age 85. The characteristic neuropathological modifications include formation of extracellular neuritic plaques and intraneuronal tangles with associated neuronal loss in hippocampus and neocortex. The major constituent of the extracellular plaques is amyloid β-protein, which aggregates into 8 nm filaments. Aβ is a peptide of 40 or 42 amino acid residues and is proteolytically derived from a transmembrane glycoprotein known as β-amyloid precursor protein.

                  The enzymes that cleave βAPP to Aβ are called as secretases, βAPP is widely expressed, particularly in brain, and its gene has been localized to chromosome 21q. Two major observations have aided in understanding the role of Aβ peptides in the pathology of Alzheimer's disease. The first is that patients with Down syndrome have trisomy 21, exhibit Aβ deposits, and develop classical features of Alzheimer's disease at age 40 years or earlier. Second, several missense mutations in βAPP have been identified in cases of autosomal dominant Alzheimer's disease. These dominant mutations in βAPP adversely affect the action of secretases either by increasing the absolute rate of Aβ excretion (N-terminal mutations) or by increasing the ratio Aβ42 to Aβ40 (C-terminal mutations). Inherited disorders of Alzheimer's disease represent less than 1% of all cases. 

                   The Aβ peptides aggregate to form β-structures leading to fibrils. The Aβ42 peptides are more neurotoxic and produce toxic effects by many interrelated mechanisms. These may involve oxidative injury, changes in intracellular Ca2+ homeostasis, cytoskeletal reorganization, and actions by cytokines. The intraneuronal tangles are clusters of long paired helical filaments that consist of the microtubule-associated protein tau. The normal function of tau protein is to stabilize microtubules in neurons by enhancing polymerization of tubulin. Normally, tau protein is soluble; however, when it is excessively phosphorylated, it turns into an insoluble filamentous polymer. The dysregulation of phosphorylation/dephosphorylation events has been attributed to an enhanced activity of certain kinases and a diminished activity of certain phosphatases. Whereas plaques are pathognomonic for AD, tangles are found in etiologically different neurological diseases. 

                    Disorders of abnormal hyperphosphorylation and aberrant aggregation of tau protein into fibrillar polymers are known as taupathies. Examples of taupathies in addition to Alzheimer's are progressive supranuclear palsy, Pick's disease, corticobasal degeneration, and frontotemporal dementias. Two other genes in addition to βAPP have been implicated in the early onset of autosomal dominant forms of Alzheimer's disease. The other two causative genes are located on chromosomes 14 and 1 and code for transmembrane proteins presenilin 1 (consisting of 467 amino acid residues) and presenilin 2 (consisting of 448 amino acid residues). These proteins are synthesized in neurons but their functions are not known. However, mutations in the presenilin genes lead to excessive production of Aβ 42 peptides. Sporadic forms of Alzheimer's disease, responsible for 90% of all cases, are complex diseases and may represent the combined action of both environmental factors and genetic traits that manifest over long time spans. 

                    Various forms of a polymorphic gene for apolipoprotein E (apo E) which is on chromosome 19 have been found to occur in higher frequency in persons with Alzheimer's disease. There are three alleles of the apo E gene with six combinations: ε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3, ε2/ε4, and ε3/ε4. Apo E is a lipid carrier protein that is primarily synthesized in the liver; however it is also synthesized in astrocytes and neurons. Of the several genotypes for apo E, the acquisition of two apo E ε4 alleles may increase the risk for Alzheimer's disease up to eight fold. Each copy of the apo E gene increases the risk and shifts the onset to lower ages. The biochemical mechanism by which apo E ε4 protein participates in formation of tangles and plaques is unclear. Several mechanisms have been suggested, namely, interaction with tau protein and generation, and clearance of Aβ peptides. Pharmacological therapy for Alzheimer's disease consists of correcting the cholinergic deficit by administering acetylcholinesterase inhibitors. Estrogen therapy in women with Alzheimer's disease has been associated with improved cognitive performance. Estrogen's beneficial effect may be due to cholinergic and neurotrophic actions. Other therapeutic strategies are directed at inhibiting or decreasing the formation of neurotoxic peptides. In addition, drugs that selectively digest the aggregated peptides may prove useful. 

                       An experimental vaccine which contains AP peptide administered to plaque-producing mice leads to less plaque formation in younger mice and the disappearance of plaques in the older mice. The alterations in the plaque formation in mice were associated with preservation of memory and learning ability. The vaccination did not trigger an autoimmune response or toxic reaction in the experimental animals. Thus, these studies have provided impetus for the development of a human vaccine.

                        In evaluating a patient for Alzheimer's disease, it is essential that other treatable causes of dementia be excluded by determining critical biochemical and clinical parameters. Some of the treatable, relatively common abnormalities that produce dementia include drug abuse, electrolyte imbalance, thyroid abnormalities, and vitamin B12 deficiency; less common abnormalities are tumor, stroke, and Wernicke 's encephalopathy.

                            Transthyretin amyloidosis  is an autosomal dominant syndrome characterized by peripheral neuropathy. This disease results from one of five mutations identified thus far in the gene for transthyretin. Transthyretin is also called prealbumin because it migrates ahead of albumin in standard electrophoresis at pH 8.6. Transthyretin is synthesized in the liver and is a normal plasma protein with a concentration of 20-40 mg/dL. It transports thyroxine and retinol binding protein. The concentration of transthyretin is
significantly decreased in malnutrition and plasma levels are diagnostic of disorders of malnutrition. The gene responsible for transthyretin expression resides on chromosome 18 and it is expressed in a constitutive manner. The primary structure of transthyretin consists of 127 amino acid residues and eight β-sheet structures arranged in an antiparallel conformation on parallel planes. Protein folding disorders of an unusual nature may account for a group of transmissible spongiform encephalopathies (TSE) involving proteins called prions. These disorders, known as prion diseases, are all characterized by amyloid deposition in the brain of animals and humans. The clinical features include neurological symptoms with loss of motor control, dementia, paralysis, and wasting. Incubation periods for prion diseases are months in animals and years in humans. No treatments are available for any of these diseases. 

                             TSEs occur in several species of animals and humans, and animal models have been essential in deciphering the molecular basis of these diseases. TSEs can exhibit inherited, infectious and sporadic presentations. Additionally, the inherited disease can also be infectious. CJD happens both as an inherited autosomal dominant disorder and in a transmissible proces. According to the protein only hypothesis, the abnormal prion protein, either introduced from external sources or produced by the mutated prion protein gene, affects normal protein folding and shifts the prion protein folding towards the formation of abnormal prion protein. The conversion of the normal prion protein, whose function is unknown, to an aberrant form involves a conformational change rather than a covalent modification. The abnormal prion protein functions
as a seed that induces the normal cellular prion protein towards the abnormal amyloidogenic rich, β-structure proteins which can be propagated and transmitted to other cells. The aggregated form of prion protein forming amyloid is resistant to proteolysis. 

                                The conversion of naturally occurring protease sensitive prion protein to a protease-resistanct form occurs in vitro by mixing the two proteins. However, these protease-resistant prion proteins are not infectious. Thus, in the "protein-only" hypothesis of prion infection, the acquisition of an abherrant conformation is not sufficient for the propagation of infectivity. However, in the yeast system, the abnormal prion form of the yeast protein, introduced by liposome fusion, is able to seed a self-propagating conformational change of the normal proteins, which accumulate as aggregates. The aggregates are transmissible to daughter yeast cells along with the propagation of abnormal phenotype. Recently a serious public health problem has arisen by showing that a prion disease in cattle can cross species barriers and infect humans. This occurred when cattle were fed meal made from sheep infected with scrapie. The cattle developed mad cow disease (BSE). Subsequently, when people consumed prion-contaminated beef, a small number, primarily in Great Britain, developed a variant of CJD (vCJD) approximately five years afterward. The variant form of CJD is a unique form of prion disease occurring in a much younger population than would be expected from inherited or sporadic CJD. Both BSE and vCJD share many similar pathologic characteristics suggesting an etiologic link between human vCJD and cattle BSE. 

                                  The tumor suppressor protein p53 provides yet another example of protein misfolding that can lead to pathological effects, in this case cancers. The gene for p53 is located on the short arm of chromosome 17 and codes for a 393-amino acid phosphoprotein. In many cancers the p53 gene is mutated and the lack of normal p53 protein has been linked to the development of as many as 40% of human cancers. Normal p53 functions as a tumor suppressor and is a transcription factor that normally participates in the regulation of several genes required to control cell growth, DNA repair, and apoptosis. Normal p53 is a tetramer and it binds to DNA in a sequence specific manner. One of the p53-regulated genes produces a protein known as p21, which interferes with the cell cycle by binding to cyclin kinases. 

                                   Other genes regulated by p53 are MDM2 and BAX. The former gene codes for a protein that inhibits the action of p53 by functioning as a part of a regulatory feedback mechanism. The protein made by the BAX gene is thought to play a role in p53-induced apoptosis. Most mutations of p53 genes are somatic missense mutations which involve amino acid substitutions in the DNA binding domain. The mutant forms of p53 are misfolded proteins with abnormal conformations and the unable to bind to DNA, or they are less stable. Individuals with the rare disorder Li-Fraumeni syndrome have one mutated p53 gene and one normal p53 gene. These individuals have increased susceptibility to many cancers, such as leukemia, breast carcinomas, soft-tissue sarcomas, brain tumors, and osteosarcomas. Clinical trials are underway to investigate whether the introduction of normal p53 gene into tumor cells by means of gene therapy has beneficial effects in the treatment of cancer. Early results with p53 gene therapy indicate that it may shrink the tumor by triggering apoptosis.

Please find the below table for examples of protein folding diseases:

Disease	Protein	Molecular Phenotype
Cystic fibrosis	CFTR	Misfolding /altered Hsp70 and calnexin interaction
Marfan syndrome	Fibrillin	Misfolding
Amyotrophic lateral sclerosis	Superoxide dismutase	Misfolding
Scurvy	Collagen	Misfolding
Maple syrup urine disease	Α-Ketoacid dehydrogenase	Misfolding/Misassembly
Cancer	p53	Misfolding/altered Hsp70 interaction
Osteogenesis imperfecta	Type I procollagen pro α	Misassembly
Scrapie/Creutzfeldt-Jakob/familial insomnia	Prion protein	Aggregation
Alzheimer’s disease	Β-Amyloid	Aggregation
Familial amyloidosis	Transthyretin/lysozyme	Aggregation
Cataracts	Crystallins	Aggregation
Familial hypercholestrerolemia	LDL receptors	Improper trafficking
Α1-Antitrypsin deficiency	Α1-Antitrypsin	Improper trafficking
Tay-Sachs disease	Β-Hexosaminidase	Improper trafficking
Retinitis pigmentosa	Rhodopsin	Improper trafficking
Leprechaunism	Insulin receptor	Improper trafficking