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).

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. CONTENT
 INTRODUCTION
 HAMLET; A MEDICAL APPROACH
 OBJECTIVES
 PLAN OF WORK
 TOOLS USED IN PROTEOLYSIS
 INPUT
 RESULT AND DISCUSSION
 CONCLUSION
 FUTURE ASPECTS
 REFERENCES

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. 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. 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. MOLECULAR STRUCTRURE HUMAN ALPHA-LACTALBUMIN

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. FORMATION AND ROLE OF HAMLET IN APOPTOSIS OF CANCER CELLS

9. OBJECTIVES
TO STUDY THE PROTEOLYSIS OF α-
LACTALBUMIN BY DIFEERENT TYPE OF
PROTEASES.
Conformational Prerequisites for α-
Lactalbumin Fibrillation

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. 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. 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.

14. 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.

15. PROTEASES ACTIVITY ON α-
LACTALBUMIN

22. Comparative analysis of aminoacids of human and bovine alpha-lactalbumin

23. 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.

24. 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.

25. 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.

26. 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.

27. THANK YOU