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TO STUDY THE PROTEOLYSIS OF α-LACTALBUMIN BY
DIFEERENT TYPE OF PROTEASES.
Conformational Prerequisites for α-Lactalbumin F...
CONTENT
 INTRODUCTION
 HAMLET; A MEDICAL APPROACH
 OBJECTIVES
 PLAN OF WORK
 TOOLS USED IN PROTEOLYSIS
 INPUT
 RESU...
INTRODUCTION
• α-Lactalbumin is the second major protein in bovine milk (2-5% of the
total protein in bovine milk).
• The ...
Contd….
• During its digestion with proteases, peptides with antibacterial and
immunostimulatory properties are formed, th...
Contd….
• Amyloid fibril formation can occur when the native structure of a protein is
destabilized, favoring formation of...
MOLECULAR STRUCTRURE HUMAN ALPHA-LACTALBUMIN
HAMLET; A MEDICAL APPROACH
• HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a
tumoricidal complex consisti...
FORMATION AND ROLE OF HAMLET IN APOPTOSIS OF CANCER CELLS
OBJECTIVES
TO STUDY THE PROTEOLYSIS OF α-
LACTALBUMIN BY DIFEERENT TYPE OF
PROTEASES.
Conformational Prerequisites for α...
PLAN OF WORK
Taken the sequence of alpha-lactalbumin
From swiss-uniprot
The sequence is cleaved by different proteases
Mai...
TOOLS USED IN PROTEOLYSIS
• ExPASy is the SIB Bioinformatics Resource Portal which provides access to
scientific databases...
INPUT
• Alpha-lactalbumin; Regulatory subunit of lactose synthase, changes the substrate specificity of
galactosyltransfer...
RESULT AND DISCUSSION
• By the using of peptide cutter we have used three proteases at low
PH ;
• Trypsin cleaves peptide ...
PROTEASES ACTIVITY ON α-
LACTALBUMIN
Comparative analysis of aminoacids of human and bovine alpha-lactalbumin
DISCUSSION
• Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein,
formed amyloid fibrils at low pH, where...
Contd….
• It is well-known that α-LA is a member of the lysozyme family of proteins.
Lysozyme and its variants have been s...
CONCLUSION
The sequence of alpha-lactalbumin taken from swiss-
uniprot and was further cleaved by different proteases
main...
FUTURE ASPECTS
The bewildering array of phenotypes and alterations
seen in cancer cells necessitates cataloguing their fea...
THANK YOU
milk is very important for our life
milk is very important for our life
milk is very important for our life
milk is very important for our life
milk is very important for our life
milk is very important for our life
milk is very important for our life
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milk is very important for our life

hamlet a medical approach for cancer treatment . alpha-lactalbumin cleaved by several proteases and got several small fragment whic will join by oleic acid and form the hamlet (human alpha-lactalbumin which is lethal for tumor cell).

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milk is very important for our life

  1. 1. TO STUDY THE PROTEOLYSIS OF α-LACTALBUMIN BY DIFEERENT TYPE OF PROTEASES. Conformational Prerequisites for α-Lactalbumin Fibrillation By; Vivek Singh M.Sc. Biotechnology University of Allahabad
  2. 2. CONTENT  INTRODUCTION  HAMLET; A MEDICAL APPROACH  OBJECTIVES  PLAN OF WORK  TOOLS USED IN PROTEOLYSIS  INPUT  RESULT AND DISCUSSION  CONCLUSION  FUTURE ASPECTS  REFERENCES
  3. 3. INTRODUCTION • α-Lactalbumin is the second major protein in bovine milk (2-5% of the total protein in bovine milk). • The human variant has several physiologic functions in the neonatal period. In the mammary gland, it participates in lactose synthesis and facilitates milk production and secretion. • α-Lactalbumin binds divalent cations (Ca2+, Zn2+) and may facilitate the absorption of essential minerals. • It provides a well-balanced supply of essential amino acids for the growing infant. • α-Lactalbumin (α-LA) is a small acidic Ca2+-binding protein involved in the regulation of lactose biosynthesis as a component of lactose synthetase.
  4. 4. Contd…. • During its digestion with proteases, peptides with antibacterial and immunostimulatory properties are formed, thereby possibly helping in the protection against infection. • Hydrolysis of LA is difficult to perform, as the compact globular structure is relatively resistant toward enzymatic proteolysis. • I have used three enzymes pepsin, trypsin, chymotrypsin by the using the peptide cutter. • A number of human diseases, known as amyloidoses, originate from the deposition of stable protein aggregates, amyloid fibrils. In each of these pathological states, a specific protein or protein fragment changes from its native soluble form into insoluble fibrils, which accumulate in a variety of organs and tissues.
  5. 5. Contd…. • Amyloid fibril formation can occur when the native structure of a protein is destabilized, favoring formation of partially folded conformations. • The binding of Zn2+ ions to Ca2+-loaded α-LA decreased protein thermal stability, caused aggregation, and increased its susceptibility to protease digestion. • Native α-LA consists of two domains: a large α-helical domain and a small β- sheet domain, which are connected by a calcium binding loop. • The α-helical domain is composed of three major K-helices (residues 5-11, 23- 24, and 86-98) and two short 310 helices (residues 18-20, and 115-118). • The small domain is composed of a series of loops, a small three-stranded antiparallel L-pleated sheet.
  6. 6. MOLECULAR STRUCTRURE HUMAN ALPHA-LACTALBUMIN
  7. 7. HAMLET; A MEDICAL APPROACH • HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. • Oleic acid binds with partially unfolded α-lactalbumin on the stationary phase in situ. • HAMLET has been shown to cause DNA damage in tumor cell lines as well as in vivo in human tumors using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay which labels the free 3′- hydroxyl DNA ends generated during DNA fragmentation caused by programmed cell death. • In the nucleus it binds to histones H3, H2B and H4 and impairs transcription .
  8. 8. FORMATION AND ROLE OF HAMLET IN APOPTOSIS OF CANCER CELLS
  9. 9. OBJECTIVES TO STUDY THE PROTEOLYSIS OF α- LACTALBUMIN BY DIFEERENT TYPE OF PROTEASES. Conformational Prerequisites for α- Lactalbumin Fibrillation
  10. 10. PLAN OF WORK Taken the sequence of alpha-lactalbumin From swiss-uniprot The sequence is cleaved by different proteases Mainly with trypsin ,chymotrypsin, pepsin Get small fragment of alpha-lactalbumin Will further study for amyloid fibril formation & HAMLET formation In simulation experiment.
  11. 11. TOOLS USED IN PROTEOLYSIS • ExPASy is the SIB Bioinformatics Resource Portal which provides access to scientific databases and software tools (i.e.,resources) in different areas of life sciences including proteomics, genomics, phylogeny, systems biology, population genetics, transcriptomics etc. • UniProtKB/Swiss-Prot; It is a high quality annotated and non-redundant protein sequence database, which brings together experimental results, computed features and scientific conclusions. • PeptideCutter predicts potential cleavage sites cleaved by proteases or chemicals in a given protein sequence. PeptideCutter returns the query sequence with the possible cleavage sites mapped on it and /or a table of cleavage site positions.
  12. 12. INPUT • Alpha-lactalbumin; Regulatory subunit of lactose synthase, changes the substrate specificity of galactosyltransferase in the mammary gland making glucose a good acceptor substrate for this enzyme. This enables LS to synthesize lactose, the major carbohydrate component of milk. In other tissues, galactosyltransferase transfers galactose onto the N-acetylglucosamine of the oligosaccharide chains in glycoproteins. • Feature key- calcium binding • Position(s)- 97-108 • Length- 12 • Molecular function; • calcium ion binding lactose synthase activity • Biological process ; • apoptotic process cell-cell signaling • defense response to bacterium • lactose biosynthetic process • signal transduction • Ligandi • Calcium, Metal-binding I have taken the sequence of human alpha-lactalbumin from swiss-prot, after taking this I will check the protease activity on this sequence.
  13. 13. RESULT AND DISCUSSION • By the using of peptide cutter we have used three proteases at low PH ; • Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. • Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond (the P1position)is a largehydrophobicaminoacid(tyrosine,tryptophan,and phenylalanine ). • Pepsin is most efficient in cleaving peptide bondsbetween hydrophobic and preferably aromatic amino acids such as phenylalanine, tryptophan, and tyrosine.
  14. 14. PROTEASES ACTIVITY ON α- LACTALBUMIN
  15. 15. Comparative analysis of aminoacids of human and bovine alpha-lactalbumin
  16. 16. DISCUSSION • Bovine alpha-lactalbumin, a small acidic Ca(2+)-binding milk protein, formed amyloid fibrils at low pH, where it adopted the classical molten globule-like conformation. • S-(Carboxymethyl)-alpha-lactalbumin, a disordered form of the protein with three out of four disulfide bridges reduced, was even more susceptible to fibrillation. Other partially folded conformations induced in the intact protein at neutral pH, either by the removal of Ca(2+) or by the binding of Zn(2+) to the Ca(2+)-protein complex, did not fibrillate, although Zn(2+)-loaded α-lactalbumin precipitated out of solution as amorphous aggregates. • The three-dimensional structure of α-LA is very similar to that of lysozyme, i.e., α-LA is comprised of a large α-helical domain and a small α-sheet domain connected by a calcium-binding loop.
  17. 17. Contd…. • It is well-known that α-LA is a member of the lysozyme family of proteins. Lysozyme and its variants have been shown to form amyloid fibrils under a variety of conditions. • A novel variant of human lysozyme (Trp64Arg) has recently been reported to cause a combination of amyloid deposition with sicca syndrome and amyloid nephropathy. • In Peptide cutter full length proteolysis is seen but in lab condition only partial proteolysis occur on SDS-PAGE.
  18. 18. CONCLUSION The sequence of alpha-lactalbumin taken from swiss- uniprot and was further cleaved by different proteases mainly trypsin, pepsin,chymoyrypsin, at low PH and got several small fragment by the help of peptide cutter. Partially unfolded protein may lead to formation of HAMLET(human alpha-lactalbumin made lethal to tumor cells). native α-lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear.
  19. 19. FUTURE ASPECTS The bewildering array of phenotypes and alterations seen in cancer cells necessitates cataloguing their feature and hallmarks .Here we consider that it is not only possible to identify the features of tumour cells that cause transformation but that it is also feasible to identify those genes and signalling pathways that can act specifically against cancer cells. It is hoped that such efforts might reveal new ‘‘Achilles heels’’ in cancer cells that can be exploited for their specific culling and the development of improved cancer treatments.
  20. 20. THANK YOU
  • nullspam

    Jan. 28, 2020
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    Aug. 18, 2017
  • viveksingh9849

    May. 17, 2017

hamlet a medical approach for cancer treatment . alpha-lactalbumin cleaved by several proteases and got several small fragment whic will join by oleic acid and form the hamlet (human alpha-lactalbumin which is lethal for tumor cell).

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